1. Field of the Invention
This invention relates to certain substituted fused tricyclic compounds containing quinolinonitrile rings as well as the pharmaceutically acceptable salts thereof. The compounds of the present invention inhibit the action of certain growth factor receptor protein tyrosine kinases (PTK) and other protein kinases thereby inhibiting the abnormal growth of certain cell types. The compounds of this invention are therefore useful for the treatment of certain diseases that are the result of deregulation of these PTKs. The compounds of this invention are anti-cancer agents and are useful for the treatment of cancer in mammals. In addition, the compounds of this invention are useful for the treatment or inhibition of polycystic kidney disease and colonic polyps in mammals. This invention also relates to the manufacture of said substituted fused tricyclic compounds, their use for the treatment of cancer and polycystic kidney disease, and the pharmaceutical preparations containing them.
2. Description of the Prior Art
Protein tyrosine kinases are a class of enzymes that catalyze the transfer of a phosphate group from ATP to a tyrosine residue located on a protein substrate. Protein tyrosine kinases clearly play a role in normal cell growth. Many of the growth factor receptor proteins function as tyrosine kinases and it is by this process that they effect signaling. The interaction of growth factors with these receptors is a necessary event in normal regulation of cell growth. However, under certain conditions, as a result of either mutation or overexpression, these receptors can become deregulated; the result of this is uncontrolled cell proliferation which can lead ti tumor growth and ultimately to the disease known as cancer [Wilks, A. F., Adv. Cancer Res., 60, 43 (1993) and Parsons, J. T.; Parsons, S. J., Important Advances in Oncology, DeVita, V. T. Ed., J. B. Lippincott Co., Phila., 3 (1993)]. Among the growth factor receptor kinases and their proto-oncogenes that have been identified and which are targets of the compounds of this invention are the epidermal growth factor receptor kinase (EGF-R kinase, the protein product of the erbB oncogene), and the product produced by the erbB-2 (also referred to as the neu or HER2) oncogene. Since the phosphorylation event is a necessary signal for cell division to occur and since overexpressed or mutated kinases have been associated with cancer, an inhibitor of this event, a protein tyrosine kinase inhibitor, will have therapeutic value for the treatment of cancer and other diseases characterized by uncontrolled or abnormal cell growth. For example, overexpression of the receptor kinase product of the erbB-2 oncogene has been associated with human breast and ovarian cancers [Slamon, D. J., et. al., Science, 244, 707 (1989) and Science, 235, 1146 (1987)]. Deregulation of EGF-R kinase has been associated with epidermoid tumors [Reiss, M., et. al., Cancer Res., 51, 6254 (1991)], breast tumors [Macias, A., et. al., Anticancer Res., 7, 459 (1987)], and tumors involving other major organs [Gullick, W. J., Brit. Med. Bull., 47, 87 (1991)]. Because of the importance of the role played by deregulated receptor kinases in the pathogenesis of cancer, many recent studies have dealt with the development of specific PTK inhibitors as potential anti-cancer therapeutic agents [some recent reviews: Garcia-Echeverria, C., et al. Med. Res. Rev. 20, 28-57 (2000) and Bridges, A. J. Current Medicinal Chemistry, 6, 825-843 (1999)]. The compounds of this invention inhibit the kinase activity of EGF-R and are therefore useful for treating certain disease states, such as cancer, that result, at least in part, from deregulation of this receptor. The compounds of this invention are also useful for the treatment and prevention of certain pre-cancerous conditions, such as the growth of colon polyps, that result, at least in part, from deregulation of this receptor.
It is also known that deregulation of EGF receptors is a factor in the growth of epithelial cysts in the disease described as polycystic kidney disease [Du, J.; Wilson, P. D., Amer. J. Phlysiol., 269(2 Pt 1), 487 (1995); Nauta, J., et al., Pediatric Research, 37(6), 755 (1995); Gattone, V. H., et al., Developmental Biology, 169(2), 504 (1995); Wilson, P. D., et al., Eur. J. Cell Biol., 61(1), 131, (1993)]. The compounds of this invention, which inhibit the catalytic function of the EGF receptors, are consequently useful for the treatment of this disease.
The mitogen-activated protein kinase (MAPK) pathway is a major pathway in the cellular signal transduction cascade from growth factors to the cell nucleus. The pathway involves kinases at two levels: MAP kinase kinases (MAPKK), and their substrates MAP kinases (MAPK). There are different isoforms in the MAP kinase family. [For review, see Seger, R.; Krebs, E. G., FASEB, 9, 726 (1995)]. The compounds of this invention can inhibit the action of two of these kinases: MEK, a MAP kinase kinase, and its substrate ERK, a MAP kinase. MEK is activated by phosphorylation on two serine residues by upstream kinases such as members of the raf family. When activated, MEK catalyzes phosphorylation on a threonine and a tyrosine residue of ERK. The activated ERK then phosphorylates and activates transcription factors in the nucleus, such as fos and jun, or other cellular targets with PXT/SP sequences. ERK, a p42 MAPK is found to be essential for cell proliferation and differentiation. Overexpression and/or over-activation of MEK or ERK has been found to be associated with various human cancers [For example, Sivaraman, V. S.; Wang, H. -Y.; Nuovo, G. J.; Malbon, C. C., J. Clin. Invest. 99, 1478 (1997)]. It has been demonstrated that inhibition of MEK prevents activation of ERK and subsequent activation of ERK substrates in cells, resulting in inhibition of cell growth stimulation and reversal of the phenotype of ras-transformed cells [Dudley, D. T.; Pang, L.; Decker, S. J.; Bridges, A. J.; Saltiel, A. R., Proc. Nat. Acad. Sci., 92, 7686, (1995)]. Since, as demonstrated below, the compounds of this invention can inhibit the coupled action of MEK and ERK , they are useful for the treatment of diseases such as cancer which are characterized by uncontrolled cell proliferation and which, at least in part, depend on the MAPK pathway.
Members of the raf family of kinases phosphorylate serine residues on MEK. There are three serine/threonine kinase members of the raf family known as a-raf, b-raf and c-raf. While mutations in the raf genes are rare in human cancers, c-raf is activated by the ras oncogene which is mutated in a wide number of human cancers. Therefore inhibition of the kinase activity of c-raf may provide a way to prevent ras mediated tumor growth [Campbell, S. L., Oncogene, 17, 1395 (1998)].
c-Met, a receptor tyrosine kinase, and its ligand, scatter factor (SF), are involved in epithelial cell proliferation and motility, and c-Met is overexpressed in a variety of neoplastic tissues. Simultaneous expression of HGF (hepatocyte growth factor) and c-Met in a non-tumor murine cell line results in transformation and metastasis in vivo [review: Jeffers, M., et al, J. of Molecular Medicine 74, 505 (1996)]. Kaji et al reported that antisense oligonucleotides which bind the start codon of c-Met, result in significantly decreased cell number of gastric carcinoma cell lines [Kaji, M., et al, Cancer Gene Therapy, 3, 393 (1996)]. The compounds of this invention can inhibit c-Met kinase, and are consequently useful for treatment of this disease.
Epithelial Cell Kinase (ECK) is a receptor protein tyrosine kinase (RPTK) belonging to the EPH (Erythropoietin Producing Hepatoma) family. Although originally identified as an epithelial lineage-specific tyrosine kinase, ECK has subsequently been shown to be expressed on vascular endothelial cells, smooth muscle cells, and fibroblasts. ECK is a type I transmembrane glycoprotein with the extracellular ligand-binding domain consisting of a cysteine-rich region followed by three fibronectin type m repeats. The intracellular domain of ECK possesses a tyrosine kinase catalytic domain that initiates a signal transduction cascade reflecting the ECK function. ECK binds and is subsequently activated by its counter-receptor, Ligand for Eph-Related Kinase (LERK)-1, which is an immediate early response gene product readily inducible in a lineage-unrestricted manner with proinflammatory cytokines such as IL-1 or TNF. Soluble LERK-1 has been shown to stimulate angiogenesis in part by stimulating ECK in a murine model of corneal angiogenesis. Unlike their normal counterparts, tumor cells of various lineages constitutively express LERK-1 and this expression can further be upregulated by hypoxia and proinflammatory cytokines. Many of these tumor cells also express ECK at higher levels than their normal counterparts, thereby creating an opportunity for autocrine stimulation via ECK: LERK-1 interaction. The increased expression of both ECK and LERK-1 has been correlated with the transformation of melanomas from the noninvasive horizontal phase of growth into very invasive vertically growing metastatic melanomas. Together, the ECK LERK-1 interaction is believed to promote tumor growth via its tumor growth promoting and angiogenic effects. Thus, the inhibition of the ECK tyrosine kinase activity mediating signaling cascade induced by its binding and cross-linking to LERK-1 may be therapeutically beneficial in cancer, inflammatory diseases, and hyperproliferative disorders.
The Src family of cytoplasmic protein tyrosine kinases consists of at least eight members (Src, Fyn, Lyn, Yes, Lck, Fgr, Hck and Blk) that participate in a variety of signaling pathways [Schwartzberg, P. L., Oncogene, 17, 1463-1468, (1998)]. The prototypical member of this tyrosine kinase family is p60src (Src). Src is involved in proliferation and migration responses in many cell types. In limited studies, Src activity has been shown to be elevated in breast, colon (xcx9c90%), pancreatic ( greater than 90%) and liver ( greater than 90%) tumors. Greatly increased Src activity is also associated with metastasis ( greater than 90%) and poor prognosis. Antisense Src message impedes growth of colon tumor cells in nude mice [Staley et al., Cell Growth and Differentiation., 8, 269-74, (1997)], suggesting that Src inhibitors should slow tumor growth. In addition to its role in cell proliferation, Src also acts in stress response pathways, including the hypoxia response, and nude mice studies with colon tumor cells expressing antisense Src message have reduced vascularization [Ellis, et al., J. Biol. Chem., 273, 1052-7 (1998)], which suggests that Src inhibitors would be anti-angiogenic as well as anti-proliferative.
Growth of most solid tumors is dependent on the angiogenesis involving activation, proliferation and migration of vascular endothelial cells and their subsequent differentiation into capillary tubes. Angiogenization of tumors allows them access to blood-derived oxygen and nutrients, and also provides them adequate perfusion. Hence inhibiting angiogenesis is an important therapeutic strategy in not only cancer but also in a number of chronic diseases such as rheumatoid arthritis, psoriasis, diabetic retinopathy, age-related macular degeneration, and so on. Tumor cells produce a number of angiogenic molecules. Vascular Endothelial Growth Factor (VEGF) is one such angiogenic factor. VEGF, a homodimeric disulfide-linked member of the PDGF family, is an endothelial cell-specific mitogen and is known to cause profound increase in the vascular endothelial permeability in the affected tissues. VEGF is also a senescence-preventing survival factor for endothelial cells. Almost all nucleated tissues in the body possess the capability to express VEGF in response to various stimuli including hypoxia, glucose deprivation, advanced glycation products, inflammatory cytokines, etc. Growth-promoting angiogenic effects of VEGF are mediated predominantly via its signaling receptor Kinase insert Domain containing Receptor (KDR). The expression of KDR is low on most endothelial cells; however, activation with angiogenic agents results in a significant upregulation of KDR on endothelial cells. Most angiogenized blood vessels express high levels of KDR. Binding to VEGF causes dimerization of KDR resulting in its autophosphorylation and initiation of a signaling cascade. Tyrosine kinase activity of KDR is essential for mediation of its functional effects as a receptor for VEGF. Inhibition of KDR-mediated functional effects by inhibiting KDR""s catalytic activity is considered to be an important therapeutic strategy in the treatment of angiogenized disease states including cancer. Normal angiogenesis is required in many physiological conditions such as wound healing, female reproduction and fetal development. Abnormal or pathological angiogenesis has been implicated in neoplastic diseases including solid tumor growth, metastasis, and Kaposi""s sarcoma; various eye diseases including diabetic retinopathy, and macular degeneration; inflammatory conditions including rheumatoid arthritis, and osteoarthritis; skin diseases including psoriasis, eczema and scleroderma; as well as ulcerative colitis and childhood haemangiomas [Toi, M. et al., Breast Cancer Res. And Treat., 36, 192-204 (1995); Folkman, J., Nature Medicine, 1, 27-31 (1995); Jackson, J. R. et al., FASEB J., 11, 457-465 (1997)]. Inhibition of VEGF function has been shown to inhibit disease progression in tumors [Borgstrom, P. et al., Cancer Res., 56, 4032-4039 (1996); Kim, J. K. et al., Nature, 362, 841-844 (1993)] and retinal neovascularization [Aiello, L. P. et al., Proc. Nat. Acad. Sci., 92, 10457-10461 (1995)] as well as vascular dysfunction mediated by glucose in models of diabetes [Tilton, R. G. et al., J. Clin. Invest., 99, 2192-2202 (1997)].
Some 3-cyano-quinoline derivatives are inhibitors of tyrosine kinases and are described in: WO-98/43960 (U.S. Pat. No. 6,002,008). The patent U.S. Pat. No. 5,780,482 and application WO-95/00511 describe some condensed 4-aminopyridine compounds that have antirheumatic activity and can contain a cyano group at the 3-position. A 3-cyano-quinoline with a 4-(2-methylanilino) substituent having gastric (H+/K+)-ATPase inhibitor activity at high concentrations has been described [Ife, R. J., et al., J. Med. Chem., 35 (18), 3413 (1992)].
Quinolines that do not have the 3-cyano substituent have been reported, and, unlike the compounds of this invention, are unsubstituted at the 4-position but are reported to be inhibitors of protein tyrosine kinases [Gazit A., et al., J. Med. Chem., 39(11), 2170 (1996)]. A series of quinolines that have a 3-pyridyl substituent and no substituent at the 4-position have been described as inhibitors of platelet derived growth factor receptor kinase [Dolle, R. E., et al., J. Med. Chem., 372, 2627 (1994) and Maguire, M. P., et al., J. Med. Chem., 372, 129 (1994)]. The patent application WO 96/09294 describes inhibitors of protein tyrosine kinases that include 4-anilino quinolines with a large variety of substituents on positions 5-8 but which must also have a hydrogen atom at position 3. An international patent WO-98/13350 describes 3-fluoroquinoline and quinoline tyrosine kinase inhibitors. The U.S. Pat. No. 5,480,883 describes quinoline derivatives that are inhibitors of protein tyrosine kinases but these derivatives do not have the the 3-cyano group.
Certain tricyclic compounds are reported in the applications WO-92/02508, EP-412848 and EP-456442 where positions 6 and 7 of the 3-cyano-quinoline are substituted with alkylene-dioxy groups forming a third ring of size 5-8, however, the only hetero atoms present in the third ring are two oxygen atoms; furthermore, only tricycles substituted with alkoxy groups at the 4-position are claimed as angiotensin II antagonists. The U.S. Pat. No. 6,002,008 describes that contiguous carbon atoms of 5-8 of 3-cyano-quinolines are substituted with, ethylen-dioxy, a divalent radical to form tricyclic compounds as inhibitors of protein tyrosine kinases, however, the only hetero atoms present in the third ring are two oxygen atoms.
The compounds of this invention are certain 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinolines, 3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinolines, 2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinolines, and 3,4-dihydro-2H-[1,4]thiazino[2,3-g]quinolines which are inhibitors of protein tyrosine kinase and are useful as antineoplastic agents.
In accordance with the present invention, there is provided compounds represented by Formula (1): 
wherein:
Z is xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94S(O)axe2x80x94, or xe2x80x94NRxe2x80x94;
R is alkyl of 1 to 6 carbon atoms, or carboalkyl of 2 to 7 carbon atoms;
X is cycloalkyl of 3 to 7 carbon atoms, which may be optionally substituted with one or more alkyl of 1 to 6 carbon atom groups; or
X is pyridinyl, pyrimidinyl, or an aryl ring; wherein the pyridinyl, pyrimidinyl or aryl ring may be optionally mono-, di-, or tri-substituted with a substituent independently selected from the group consisiting of halogen, oxo, thio, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, azido, hydroxyalkyl of 1 to 6 carbon atoms, halomethyl, alkoxymethyl of 2 to 7 carbon atoms, alkanoyloxymethyl of 2 to 7 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkylthio of 1 to 6 carbon atoms, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy of 2 to 7 carbon atoms, carboalkyl of 2 to 7 carbon atoms, phenoxy, phenyl, thiophenoxy, benzoyl, benzyl, amino, alkylamino of 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, phenylamino, benzylamino, alkanoylamino of 1 to 6 carbon atoms, alkenoylamino of 3 to 8 carbon atoms, alkynoylamino of 3 to 8 carbon atoms, carboxyalkyl of 2 to 7 carbon atoms, carboalkoxyalkyl of 3 to 8 carbon atoms, aminoalkyl of 1 to 5 carbon atoms, N-alkylaminoalkyl of 2 to 9 carbon atoms, N,N-dialkylaminoalkyl of 3 to 10 carbon atoms, N-alkylaminoalkoxy of 2 to 9 carbon atoms, N,N-dialkylaminoalkoxy of 3 to 10 carbon atoms, mercapto, methylmercapto, and benzoylamino; or
X is a bicyclic aryl or bicyclic heteroaryl ring system of 8 to 12 atoms, where the bicyclic heteroaryl ring contains 1 to 4 heteroatoms independently selected from N, O, and S; wherein the bicyclic aryl or bicyclic heteroaryl ring may be optionally mono-, di-, tri-, or tetra-substituted with a substituent independently selected from the group consisting of halogen, oxo, thio, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, azido, hydroxyalkyl of 1 to 6 carbon atoms, halomethyl, alkoxymethyl of 2 to 7 carbon atoms, alkanoyloxymethyl of 2 to 7 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkylthio of I to 6 carbon atoms, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy of 2 to 7 carbon atoms, carboalkyl of 2 to 7 carbon atoms, phenoxy, phenyl, thiophenoxy, benzoyl, benzyl, amino, alkylamino of 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, phenylamino, benzylamino, alkanoylamino of 1 to 6 carbon atoms, alkenoylamino of 3 to 8 carbon atoms, alkynoylamiino of 3 to 8 carbon atoms, carboxyalkyl of 2 to 7 carbon atoms, caiboalkoxyalkyl of 3 to 8 carbon atoms, aminoalkyl of 1 to 5 carbon atoms, N-alkylaminoalkyl of 2 to 9 carbon atoms, N,N-dialkylaminoalkyl of 3 to 10 carbon atoms, N-alkylaminoalkoxy of 2 to 9 carbon atoms, N,N-dialkylaminoalkoxy of 3 to 10 carbon atoms, mercapto, methylmercapto, and benzoylamino; or
X is the radical 
E is pyridinyl, pyrimidinyl, or an aryl ring; wherein the pyridinyl, pyrimidinyl or aryl ring may be optionally mono-, di-, or tri-substituted with a substituent selected from the group consisiting of halogen, oxo, thio, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, azido, hydroxyalkyl of 1 to 6 carbon atoms, halomethyl, alkoxymethyl of 2 to 7 carbon atoms, alkanoyloxymethyl of 2 to 7 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkylthio of 1 to 6 carbon atoms, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy of 2 to 7 carbon atoms, carboalkyl of 2 to 7 carbon atoms, phenoxy, phenyl, thiophenoxy, benzoyl, benzyl, amino, alkylamino of 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, phenylamino, benzylamino, alkanoylamino of 1 to 6 carbon atoms, alkenoylamino of 3 to 8 carbon atoms, alkynoylamino of 3 to 8 carbon atoms, carboxyalkyl of 2 to 7 carbon atoms, carboalkoxyalkyl of 3 to 8 carbon atoms, aminoalkyl of 1 to 5 carbon atoms, N-alkylaminoalkyl of 2 to 9 carbon atoms, N,N-dialkylaminoalkyl of 3 to 10 carbon atoms, N-alkylaminoalkoxy of 2 to 9 carbon atoms, N,N-dialkylaminoalkoxy of 3 to 10 carbon atoms, mercapto, methylmercapto, and benzoylamino;
T is substituted on E at a carbon and is
xe2x80x94NH(CH2)mxe2x80x94, xe2x80x94O(CH2)mxe2x80x94, xe2x80x94S(O)axe2x80x94(CH2)mxe2x80x94, xe2x80x94NR(CH2)mxe2x80x94, xe2x80x94(CH2)mxe2x80x94
xe2x80x94(CH2)mNHxe2x80x94, xe2x80x94(CH2)mOxe2x80x94, xe2x80x94(CH2)mS(O)axe2x80x94, or xe2x80x94(CH2)mNR xe2x80x94;
L is an aryl ring; that is optionally mono-, di, or tri-substituted with a substituent selected from the group consisting of halogen, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, azido, hydroxyalkyl of 1 to 6 carbon atoms, halomethyl, alkoxymethyl of 2 to 7 carbon atoms, alkanoyloxymethyl of 2 to 7 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkylthio of 1 to 6 carbon atoms, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy of 2 to 7 carbon atoms, carboalkyl of 2 to 7 carbon atoms, phenoxy, phenyl, thiophenoxy, benzoyl, benzyl, amino, alkylamino of 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, phenylamino, benzylamino, alkanoylamino of 1 to 6 carbon atoms, alkenoylamino of 3 to 8 carbon atoms, alkynoylamino of 3 to 8 carbon atoms, carboxyalkyl of 2 to 7 carbon atoms, carboalkoxyalkyl of 3 to 8 carbon atoms, aminoalkyl of 1 to 5 carbon atoms, N-alkylaminoalkyl of 2 to 9 carbon atoms, N,N-dialkylaminoalkyl of 3 to 10 carbon atoms, N-alkylaminoalkoxy of 2 to 9 carbon atoms, N,N-dialkylaminoalkoxy of 3 to 10 carbon atoms, mercapto, methylmercapto, and benzoylamino; or
L is a 5- or 6-membered heteroaryl ring where the heteroaryl ring contains 1 to 3 heteroatoms independently selected from N, O, and S; wherein the heteroaryl ring may be optionally mono- or di-substituted with a substituent selected from the group consisting of halogen, oxo, thio, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, azido, hydroxyalkyl of 1 to 6 carbon atoms, halomethyl, alkoxymethyl of 2 to 7 carbon atoms, alkanoyloxymethyl of 2 to 7 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkylthio of 1 to 6 carbon atoms, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy of 2 to 7 carbon atoms, carboalkyl of 2 to 7 carbon atoms, phenoxy, phenyl, thiophenoxy, benzoyl, benzyl, amino, alkylamino of 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, phenylamino, benzylamino, alkanoylamino of 1 to 6 carbon atoms, alkenoylamino of 3 to 8 carbon atoms, alkynoylamino of 3 to 8 carbon atoms, carboxyalkyl of 2 to 7 carbon atoms, carboalkoxyalkyl of 3 to 8 carbon atoms, aminoalkyl of 1 to 5 carbon atoms, N-alkylaminoalkyl of 2 to 9 carbon atoms, N,N-dialkylaminoalkyl of 3 to 10 carbon atoms, N-alkylaminoalkoxy of 2 to 9 carbon atoms, N,N-dialkylaminoalkoxy of 3 to 10 carbon atoms, mercapto, methylmercapto, and benzoylamino;
Axe2x80x3 is a moiety selected from the group 
G1, G2, G3 and G4 are independently selected from the group consisting of hydrogen, and alkyl of 1 to 6 carbon atoms;
R1 is xe2x80x94H, R11xe2x80x94CH2xe2x80x94, xe2x80x94R12, 
R11 is xe2x80x94H, alkyl of 1 to 5 carbon atoms, aryl, or R13xe2x80x94(C(R6)2)kxe2x80x94;
R13 is 
R7xe2x80x94, R7xe2x80x94(C(R6)2)pxe2x80x94Mxe2x80x94, or Het-(C(R6)2)qxe2x80x94Wxe2x80x94;
R7 is xe2x80x94H, xe2x80x94NR6R6, xe2x80x94OR6, xe2x80x94J, xe2x80x94N(R6)3+, or xe2x80x94NR6(OR6); 
xe2x80x83or a bond;
Het is a heterocyclic radical selected from the group consisting of morpholine, thiomoipholine, thiomorpholine S-oxide, thiomorpholine S,S-dioxide, piperidine, pyrrolidine, aziridine, pyridine, imidazole, 1,2,3-triazole, 1,2,4-triazole, thiazole, thiazolidine , tetrazole, piperazine, furan, thiophene, tetrahydrothiophene, tetrahydrofuran, dioxane, 1,3-dioxolane, tetrahydropyran, and 
optionally mono- or di-substituted on carbon by xe2x80x94R6, hydroxy, xe2x80x94N(R6)2, xe2x80x94OR6, xe2x80x94(C(R6)2)sOR6, or xe2x80x94(C(R6)2)sN(R6)2; or
optionally monoxe2x80x94substituted on nitrogen with xe2x80x94R6; and
optionally mono or dixe2x80x94substituted on a saturated carbon with divalent radicals xe2x80x94Oxe2x80x94, or xe2x80x94O(C(R6)2)sOxe2x80x94;
R6 is independently selected from xe2x80x94H, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, carboalkyl of 2 to 7 carbon atoms, carboxyalkyl of 2 to 7 carbon atoms, phenyl, or phenyl optionally substituted with one or more halogen, alkoxy of 1 to 6 carbon atoms, trifluoromethyl, amino, alkylamino of 1 to 3 carbon atoms, dialkylamino of 2 to 6 carbon atoms, nitro, cyano, azido, halomethyl, alkoxymethyl of 2 to 7 carbon atoms, alkanoyloxymethyl of 2 to 7 carbon atoms, alkylthio of 1 to 6 carbon atoms, hydroxy, carboxyl, carboalkoxy of 2 to 7 carbon atoms, phenoxy, phenyl, thiophenoxy, benzoyl, benzyl, phenylamino, benzylamino, alkanoylamino of 1 to 6 carbon atoms, and alkyl of 1 to 6 carbon atoms;
R12 is alkylsulphonyl of 1 to 6 carbon atoms, carboalkoxy of 2 to 7 carbon atoms, or carboalkyl of 2 to 7 carbon atoms;
R5 is hydrogen, alkyl of 1 to 6 carbon atoms, carboxy, carboalkoxy of 1 to 6 carbon atoms, aryl, carboalkyl of 2 to 7 carbon atoms, 
R7xe2x80x94(C(R6)2)sxe2x80x94, R7xe2x80x94(C(R6)2)pxe2x80x94Mxe2x80x94(C(R6)2)rxe2x80x94,
R8R9xe2x80x94CHxe2x80x94Mxe2x80x94(C(R6)2)rxe2x80x94, or Het-(C(R6)2)qxe2x80x94Wxe2x80x94(C(R6)2)rxe2x80x94;
R8, and R9 are each, independently, xe2x80x94(C(R6)2)rNR6R6, or xe2x80x94(C(R6)2)rOR6;
J is independently xe2x80x94H, xe2x80x94F, or xe2x80x94Jxe2x80x2;
Jxe2x80x2 is independently chlorine, bromine, iodine, tosylate (p-toluenesulfonate), or mesylate (methanesulfonate);
Q is Qxe2x80x2, alkoxy of 1 to 6 carbon atoms, hydroxy, or hydrogen;
Qxe2x80x2 is alkyl of 1 to 6 carbon atoms;
R2 is selected from the group consisting of 
R3 is independently selected from xe2x80x94H, alkyl of 1 to 6 carbon atoms, carboxy, carboalkoxy of 1 to 6 carbon atoms, aryl, carboalkyl of 2 to 7 carbon atoms, and R3axe2x80x94(C(R6)2)sxe2x80x94;
R3a is 
R7xe2x80x94, R7xe2x80x94(C(R6)2)pxe2x80x94Mxe2x80x94,
R8R9xe2x80x94CHxe2x80x94Mxe2x80x94, or Het-(C(R6)2)qxe2x80x94Wxe2x80x94;
a is 0 to 2;
k is 1, 3to 5;
n is 0 to 1;
m is 0 to 3;
p is 2 to 4;
q is 0 to 4;
r is 1 to 4;
s is 1 to 6;
u is 0 to 4 and v is 0 to 4, wherein the sum of u+v is 2 to 4;
provided that:
a. when xe2x80x94R6 is alkenyl of 2 to 7 carbon atoms or alkynyl of 2 to 7 carbon atoms, said alkenyl of 2 to 7 carbon atoms or alkynyl of 2 to 7 carbon atoms is bound to a nitrogen or oxygen atom through a saturated carbon atom;
b. when R3 is bound to sulfur, R3 is not xe2x80x94H, carboxy, carboalkoxy, or carboalkyl;
c. when M is xe2x80x94Oxe2x80x94, and R7 is xe2x80x94OR6, then p is 1 to 4;
d. when M is xe2x80x94Oxe2x80x94, then k is 1 to 5;
e. when W is xe2x80x94Oxe2x80x94, then k is 1 to 5;
f. when R7 is xe2x80x94OR6, then k is 1 to 5;
g. when W is not a bond with Het bonded through a nitrogen atom, q is 2 to 4, and when W is a bond, q is 0;
or a pharmaceutically acceptable salt thereof.
The pharmaceutically acceptable salts are those derived from such organic and inorganic acids as: acetic, lactic, citric, tartaric, succinic, maleic, malonic, gluconic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, and similarly known acceptable acids.
Throughout this patent application, the quinoline and tricyclic ring systems will be numbered as indicated in the formulas below. 
Preferred bicyclic aryl or bicyclic heteroaryl ring systems include naphthalene, 1,2,3,4-tetrahydronaphthalene, indan, 1-oxo-indane, 1,2,3,4-tetrahydroquinoline, naphthyridine, benzofuran, 3-oxo-1,3-dihydro-isobenzofuran, benzothiaphene, 1,1-dioxo-benzothiaphene, indole, 2,3-dihydroindole, 1,3-dioxo-2,3-dihydro-1H-isoindole, benzotriazole, 1H-indazole, indoline, benzopyrazole, 1,3-benzodioxole, benzooxazole, purine, phthalimide, coumarin, chromone, quinoline, terahydroquinoline, isoquinoline, benzimidazole, quinazoline, pyrido[2,3-b]pyridine, pyrido[3,4-b]pyrazine, pyrido[3,2-c]pyridazine, pyrido[3,4-b]pyridine, 1H-pyrazole[3,4-d]pyrimidine, 1,4-benzodioxane, pteridine, 2(1H)-quinolone, 1(2H)-isoquinolone, 2-oxo-2,3-dihydro-benzthiazole, 1,2-methylenedioxybenzene, 2-oxindole, 1,4-benzisoxazine, benzothiazole, quinoxaline, quinoline-N-oxide, isoquinoline-N-oxide, quinoxaline-N-oxide, quinazoline-N-oxide, benzoxazine, phthalazine, 1,4-dioxo-1,2,3,4-tetrahydro-phthalazine, 2-oxo-1,2-dihydro-quinoline, 2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazine, 2,5-dioxo-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepine, or cinnoline.
When L is a 5 or 6-membered heteroaryl ring, preferred heteroaryl rings include pyridine, pyrimidine, imidazole, thiazole, thiazolidine, pyrrole, furan, thiophene, oxazole, or 1,2,4-triazole.
Either or both rings of the bicyclic aryl or bicyclic heteroaryl group may be fully unsaturated, partially saturated, or fully saturated. An oxo substituent on the bicyclic aryl or bicyclic heteroaryl moiety means that one of the carbon atoms is substituted with a carbonyl group. A thio substituent on the bicyclic aryl or bicyclic heteroaryl moiety means that one of the carbon atoms is substituted with a thiocarbonyl group. When a compound of this invention contains a moiety which contains a heteroaryl ring, such heteroaryl ring does not contain Oxe2x80x94O, Sxe2x80x94S, or Sxe2x80x94O bonds in the ring.
When L is a 5 or 6-membered heteroaryl ring, it may be fully unsaturated, partially saturated, or fully saturated. The heteroaryl ring may be bound to T via carbon or nitrogen. An oxo substituent on the heteroaryl ring means that one of the carbon atoms is substituted with a carbonyl group. A thio substituent on the heteroaryl ring means that one of the carbon atoms is substituted with a thiocarbonyl group.
The alkyl portion of the alkyl, alkoxy, alkanoyloxy, alkoxymethyl, alkanoyloxymethyl, alkylsulphinyl, alkylsulphonyl, alkylsulfonamido, carboalkoxy, carboalkyl, carboxyalkyl, carboalkoxyalkyl, alkanoylamino, N-alkylcarbamoyl, N,N-dialkylcarbamoyl , N-alkylaminoalkoxy, and N,N-dialkylaminoalkoxy include both straight chain as well as branched carbon chains of 1 to 7 carbon atoms. The alkenyl portion of the alkenyl, alkenoyloxymethyl, alkenyloxy, alkenylsulfonamido, substituents include both straight chain as well as branched carbon chains of 1 to 7 carbon atoms and one or more sites of unsaturation and all possible configurational isomers. The alkynyl portion of the alkynyl, alkynoyloxymethyl, alkynylsulfonamido, alkynyloxy, substituents include both straight chain as well as branched carbon chains of 2 to 7 carbon atoms and one or more sites of unsaturation. Carboxy is defined as a xe2x80x94CO2H radical. Carboalkoxy of 2 to 7 carbon atoms is defined as a xe2x80x94CO2Rxe2x80x3 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms. Carboxyalkyl is defined as a HO2Cxe2x80x94Rxe2x80x2xe2x80x3xe2x80x94 radical where Rxe2x80x2xe2x80x3 is a divalent alkyl radical of 1 to 6 carbon atoms. Carboalkoxyalkyl is defined as a Rxe2x80x3O2Cxe2x80x94Rxe2x80x2xe2x80x3xe2x80x94 radical where Rxe2x80x2xe2x80x3 is a divalent alkyl radical and where Rxe2x80x3 and Rxe2x80x2xe2x80x3 together have 2 to 7 carbon atoms. Carboalkyl is defined as a xe2x80x94CORxe2x80x3 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms. Alkanoyloxy is defined as a xe2x80x94OCORxe2x80x3 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms. Alkanoyloxymethyl is defined as Rxe2x80x3CO2CH2xe2x80x94 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms. Alkoxymethyl is defined as Rxe2x80x3OCH2xe2x80x94 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms. Alkylsulphinyl is defined as Rxe2x80x3SOxe2x80x94 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms. Alkylsulphonyl is defined as Rxe2x80x3SO2xe2x80x94 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms. Alkylsulfonamido, alkenylsulfonamido, alkynylsulfonamido are defined as Rxe2x80x3SO2NHxe2x80x94 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms, an alkenyl radical of 2 to 6 carbon atoms, or an alkynyl radical of 2 to 6 carbon atoms, respectively. N-alkylcarbamoyl is defined as Rxe2x80x3NHCOxe2x80x94 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms. N,N-dialkylcarbamoyl is defined as Rxe2x80x3 Rxe2x80x2NCOxe2x80x94 radical, where Rxe2x80x3 is an alkyl radical of 1 to 6 carbon atoms, Rxe2x80x2 is an alkyl radical of 1 to 6 carbon atoms and Rxe2x80x2, and Rxe2x80x3 may be the same or different . When X is substituted, it is preferred that it is mono-, di-, or tri-substituted, with mono- and di-substituted being most preferred. A preferred embodiment of this invention is that G1, G2, G3, and G4, are hydrogen. It is also preferred that X is a phenyl ring, Z is xe2x80x94NHxe2x80x94, and n=0.
Het is a heterocyclic radical selected from the group consisting of morpholine, thiomorpholine, thiomorpholine S-oxide, thiomorpholine S,S-dioxide, piperidine, pyrrolidine, azinidine, pyridine, imidazole, 1,2,3-triazole, 1,2,4-triazole, thiazole, thiazolidine , tetrazole, piperazine, furan, thiophene, tetrahydrothiophene, tetrahydrofuran, dioxane, 1,3-dioxolane, tetrahydropyran, and 
which may be optionally mono- or disubstituted on carbon with R6, optionally mono-substituted on nitrogen with R6, optionally mono- or di-substituted on carbon with hydroxy, xe2x80x94N(R6)2, or xe2x80x94OR6, optionally mono or di-substituted on carbon with xe2x80x94(C(R6)2)sOR6 or xe2x80x94(C(R6)2)sN(R6)2, and optionally mono or di-substituted on a saturated carbon with divalent xe2x80x94Oxe2x80x94 or xe2x80x94O(C(R6)2)sOxe2x80x94 (carbonyl and ketal groups, respectively); in some cases when Het is substituted with xe2x80x94Oxe2x80x94 (carbonyl), the carbonyl group can be hydrated. Het may be bonded to W when q=0 via a carbon atom on the heterocyclic ring, or when Het is a nitrogen containing heterocycle which also contains a saturated carbon-nitrogen bond, such heterocycle may be bonded to carbon, via the nitrogen when W is a bond. When q=0 and Het is a nitrogen containing heterocycle which also contains an unsaturated carbon-nitrogen bond, that nitrogen atom of the heterocycle may be bonded to carbon when W is a bond and the resulting heterocycle will bear a positive charge. When Het is substituted with R6, such substitution may be on a ring carbon, or in the case of a nitrogen containing heterocycle, which also contains a saturated carbon-nitrogen, such nitrogen may be substituted with R6 or in the case of a nitrogen containing heterocycle, which also contains an unsaturated carbon-nitrogen, such nitrogen may be substituted with R6 in with case the heterocycle will bear a positive charge. Preferred heterocycles include pyridine, 2,6-disubstituted morpholine, 2,5-disubstituted thiomorpholine, 2-substituted imidazole, substituted thiazole, N-substituted imidazole, N-subsitituted 1,4-piperazine, N-substituted piperadine, and N-substituted pyrrolidine.
Aryl is a phenyl ring which may be optionally mono-, di-, or tri-substituted with a substituent independently selected from the group consisiting of halogen, alkyl of 1 to 6 carbon atoms, alkenyl of 2 to 6 carbon atoms, alkynyl of 2 to 6 carbon atoms, azido, hydroxyalkyl of 1 to 6 carbon atoms, halomethyl, alkoxymethyl of 2 to 7 carbon atoms, alkanoyloxymethyl of 2 to 7 carbon atoms, alkoxy of 1 to 6 carbon atoms, alkylthio of 1 to 6 carbon atoms, hydroxy, trifluoromethyl, cyano, nitro, carboxy, carboalkoxy of 2 to 7 carbon atoms, carboalkyl of 2 to 7 carbon atoms, phenoxy, phenyl, thiophenoxy, benzoyl, benzyl, amino, alkylamino of 1 to 6 carbon atoms, dialkylamino of 2 to 12 carbon atoms, phenylamino, benzylamino, alkanoylamino of 1 to 6 carbon atoms, alkenoylamino of 3 to 8 carbon atoms, alkynoylamino of 3 to 8 carbon atoms, carboxyalkyl of 2 to 7 carbon atoms, carboalkoxyalkyl of 3 to 8 carbon atoms, aminoalkyl of 1 to 5 carbon atoms, N-alkylaminoalkyl of 2 to 9 carbon atoms, N,N-dialkylaminoalkyl of 3 to 10 carbon atoms, N-alkylaminoalkoxy of 2 to 9 carbon atoms, N,N-dialkylaminoalkoxy of 3 to 10 carbon atoms, mercapto, methylmercapto, and benzoylamino.
Phenyl as used herein refers to a 6-membered aromatic ring optionally mono, di or tri-substituted.
The compounds of this invention may contain one or more asymmetric carbon atoms; in such cases, the compounds of this invention include the individual diasteromers, the racemates, and the individual R and S entantiomers thereof. Some of the compounds of this invention may contain one or more double bonds; in such cases, the compounds of this invention include each of the possible configurational isomers as well as mixtures of these isomers. When a compound of this invention contains a moiety containing the same substituent more than once (for example, when R7 is xe2x80x94NR6R6), each substituent (R6, in this example) may be the same or different.
Preferred compounds of this invention are described below. Except as otherwise indicated below, the substituents are as defined above.
A. Compounds according to Formula (1), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
or a pharmaceutically acceptable salt thereof.
B. Compounds according to Formula (I), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
or a pharmaceutically acceptable salt thereof.
C. Compounds according to Formula (I) wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
or a pharmaceutically acceptable salt thereof.
D. Compounds according to Formula (I), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
or a pharmaceutically acceptable salt thereof.
E. Compounds according to Formula (I), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
G1, G2, G3, and G4 are H;
or a pharmaceutically acceptable salt thereof.
F. Compounds according to Formula (I) wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
G1, G2, G3, and G4 are H;
or a pharmaceutically acceptable salt thereof.
G. Compounds according to Formula (I) wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
G1, G2, G3, and G4 are H;
or a pharmaceutically acceptable salt thereof.
H. Compounds according to Formula (I), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
G1, G2, G3, and G4 are H;
or a pharmaceutically acceptable salt thereof.
I. Compounds according to Formula (1), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
R1 is selected from H, R2C(O)xe2x80x94 and R11xe2x80x94CH2xe2x80x94;
or a pharmaceutically acceptable salt thereof.
J. Compounds according to Formula (I) wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
R1 is selected from H, R2C(O)xe2x80x94 and R11xe2x80x94CH2xe2x80x94;
or a pharmaceutically acceptable salt thereof.
K. Compounds according to Formula (I) wherein Z is xe2x80x94NIxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
R1 is selected from H, R2C(O)xe2x80x94 and R11xe2x80x94CH2xe2x80x94;
or a pharmaceutically acceptable salt thereof.
L. Compounds according to Formula (I), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
R1 is selected from H, R2C(O)xe2x80x94 and R11xe2x80x94CH2xe2x80x94;
or a pharmaceutically acceptable salt thereof.
M. Compounds according to Formula (I), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
R1 is selected from H, R2C(O)xe2x80x94 and R11xe2x80x94CH2xe2x80x94;
G1, G2, G3, and G4 are H;
or a pharmaceutically acceptable salt thereof.
N. Compounds according to the Formula (I) wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
R1 is selected from H, R2C(O)xe2x80x94 and R11xe2x80x94CH2xe2x80x94;
G1, G2, G3, and G4 are H;
or a pharmaceutically acceptable salt thereof.
O. Compounds according to Formula (I) wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
R1 is selected from H, R2C(O)xe2x80x94 and R11xe2x80x94CH2xe2x80x94;
G1, G2, G3, and G4 are H;
or a pharmaceutically acceptable salt thereof.
P. Compounds according to Formula (I), wherein Z is xe2x80x94NHxe2x80x94, n is 0, X is aryl and Axe2x80x3 is the moiety 
R1 is selected from H, R2C(O)xe2x80x94 and R11xe2x80x94CH2xe2x80x94;
G1, G2, G3, and G4 are H;
or a pharmaceutically acceptable salt thereof.
Specifically preferred compounds of this invention include:
9-(3-Chloro-4-fluoroanilino)-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitrile,
1-[(2E)-4-Chloro-2-butenoyl]-9-(3-chloro-4-fluoroanilino)-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitrile,
1-[(2E)-4-Bromo-2-butenoyl ]-9-(3-chloro-4-fluoroanilino)-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitrile,
9-(3-Chloro-4-fluoroanilino)-1-[(2E)-4-(dimethylamino)-2-butenoyl]-2,3-dihydro-1-H-[1-[1,4]oxazino(3,2-g]quinoline-8-carbonitnile,
9-(3-Chloro-4-fluoroanilino)-1-[4-(dimethylamino)butanoyl]-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitrile,
1-(4-Chlorobutyl)-9-(3-chloro-4-fluoroanilino)-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitrile,
9-(3-Chloro-4-fluoroanilino)-1-[4-(dimethylamino)butyl]-2,3-dihydro-1H-[1,4]oxazino[3,2-gjquinoline-8-carbonitrile,
9-(2,4-Dichloroanilino)-3,4-di hydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitrile,
4-(4-Chlorobutyl)-9-(2,4-dichloroanilino)-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitrile, and
9-(2,4-Dichloroanilino)-4-[4-(4-ethyl-1-piperazinyl)butyl]-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitrile,
or a pharmaceutically acceptable salt thereof.
The compounds of this invention may be prepared from commercially available starting materials or starting materials which may be prepared using literature procedures. More specifically, the preparation of compounds and intermediates of this invention, 4-substituted-quinoline-3-carbonitriles 9 is described below in Flowsheet 1 where X, n, and Z are as hereinbefore described. The condensation of substituted-anilines 2 and 2-cyano-3-ethoxy-acrylic acid ethyl ester 3 by heating in the absence of solvent gives esters 4, wherein R4 is a methyl or an ethyl group. Thermal cyclization of esters 4 in refluxing 3:1 diphenyl ether/biphenyl or diphenyl ether gives 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 5, which may also exist in the 4-hydroxy-quinoline tautomeric form. Nitration of 4-oxo-1,4-dihydro-qLlinoline-3-carbonitriles 5 in trifluoroacetic acid (TFA) with ammonium nitrate at room temperature gives the nitro compounds 6. Nitro compounds 6 are refluxed with chlorinating reagent selected from the group consisting of phosphorous oxychloride, oxalyl chloride, phosphorous oxychloride and phosphorus pentachloride to furnish 4-chloro compounds 7. Condensation of 4-chloro compounds 7 with various amines, anilines, alcohols, phenols, mercaptans, and thiophenols of the Formula HZxe2x80x94(CH2)nxe2x80x94X 7a where Z, X and n are hereinbefore defined gives 4-substituted-6-nitro-quinoline-3-carbonitriles 8. This condensation may be accelerated by heating the reaction mixture together with one equivalent of pyridine hydrochloride in alcohol solvents which include isopropanol and 2-ethoxyethanol or by using bases such as trialkylamines, sodium hydride in an inert solvent which includes tetrahydrofuran (THF), sodium or potassium alkoxides in alcohol solvents which includes ethanol, and the like. Reduction of 4-substituted-6-nitro-quinoline-3-carbonitriiles 8 with iron and ammonium chloride in refluxing methanol and water furnishes 4-substituted-7-alkoxy-6-amino-quinoline-3-carbonitriles 9. Alternatively, reagents such as tin chloride and the like may be used. 
The 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 5 may be prepared by an alternate route as described below in Flowsheet 2. Reaction of anthranilic acids 10 where R4 is hereinbefore defined with dimethylformamide dimethylacetal (DMF-dimethyl acetal), with or without an inert solvent, gives intermediate esters 11. The reaction of intermediate esters 11 with the lithium anion of acetonitrile prepared by using a base which includes n-butyl lithium (n-BuLi) or the like in an inert solvent which includes tetrahydrofuran (THF) gives 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 5. 
Compounds of Formula 9, which are important intermediates for the preparation of compounds of this invention, are prepared by an alternate route as described below in Flowsheet 3 where R4, Z, n and X are hereinbefore defined. Acetylation of nitroanilines 12 with acetic anhydride (Ac2O) and water at room temperature gives nitro compounds 13. Reduction of nitro compounds 13 with iron and ammonium chloride in refluxing methanol and water furnishes anilines 14. The condensation of anilines 14 and 2-cyano-3-ethoxy-acrylic acid ethyl ester 3 by heating in the absence of solvent gives esters 15. Thermal cyclization of esters 15 in refluxing 3:1 diphenyl ether/biphenyl or diphenyl ether gives 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 16. Chlorination of 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 16 in refluxing chlorinating reagent selected from phosphorous oxychloride and oxalyl chloride furnishes 4-chloro-quinolines 17. Condensation of 4-chloro-quinolines 17 with various amines, anilines, alcohols, phenols, mercaptans, and thiophenols of the Formnula HZxe2x80x94(CH2)nxe2x80x94X 7a where Z, X and n are hereinbefore defined gives 4-substituted-6-acetamido-quinoline-3-carbonitriles 18. The condensation may be accelerated by heating the reaction mixture together with one equivalent of pyridine hydrochloride in alcohol solvents which include isopropanol and 2-ethoxyethanol or by using bases such as trialkylamines, sodium hydride in an ineil solvent which includes tetrahydrofuran (THF), sodium or potassium alkoxides in alcohol solvents which includes ethanol, and the like. Hydrolysis of 4-substituted-6-acetamido-quinoline-3-carbonitniles 18 in aqueous hydrochloric acid gives 4-substituted-6-amino-quinoline-3-carbonitiiles 9 where R4, Z, n and X are hereinbefore defined. 
The prepartion of the compounds of this invention encompassed by compounds of Formula 26, which are important intermediates for the preparation of compounds of this invention, is described below in Flowsheet 4 where R4, Z, n and X are hereinbefore defined. Acetylation of nitroanilines 19 with acetic anhydride (Ac2O) and water at room temperature gives nitro compounds 20. Reduction of nitro compounds 20 with iron and ammonium chloride in refluxing methanol and water furnishes anilines 21. The condensation of anilines 21 and 2-cyano-3-ethoxy-acrylic acid ethyl ester 3 by heating in the absence of solvent gives esters 22. Thermal cyclization of esters 22 in refluxing 3:1 diphenyl ether/biphenyl or diphenyl ether gives 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 23. Chlorination of 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 23 in refluxing chlorinating reagent selected from phosphorous oxychloride and oxalyl chloride gives 4-chloro-quinolines 24. Condensation of 4-chloro-quinolines 24 with various amines, anilines, alcohols, phenols, mercaptans, and thiophenols of Formula HZxe2x80x94(CH2)nxe2x80x94X 7a where Z, X and n are hereinbefore defined gives 4-substituted-quinoline-3-carbonitriles 25. This condensation may be accelerated by heating the reaction mixture together with one equivalent of pyridine hydrochloride in alcohol solvents which include isopropanol and 2-ethoxyethanol or by using bases which includes trialkylamines, sodium hydride in an inert solvent which includes tetrahydrofuran (THF), and the like, sodium or potassium alkoxides in alcohol solvents which includes ethanol, and the like. Hydrolysis of 4-substituted-7-acetamido-quinoline-3-carbonitriles 25 in aqueous hydrochloric acid gives 4-substituted-7-amino-quinoline-3-caibonitriles 26. 
The preparation of the compounds and interrnediates of this invention encompassed by Formula 29 is described below in Flowsheet 5 where R4, Z, n, X, G1, G2, G3, and G4 are hereinbefore defined. Dealkylation of 4-substituted-7-alkoxy-6-amino-quinoline-3-carbonitriles 9 is accomplished by heating with excess amount of pyridinium hydrochloride to give 4-substituted-6-amino-7-to hydroxy-quinoline-3-carbonitriles 27. In those cases where X of 4-substituted-7-alkoxy-6-amino-quinoline-3-carbonitriles 9 is an aryl, heteroaryl, bicyclic aryl or bicyclic heteroaryl which is substituted with a methoxy or an ethoxy group, the product 4-substituted-6-amino-7-hydroxy-quinoline-3-carbonitriles 27 may be a mixture containing mono-dealkylated and di-dealkylated compounds. The mixture may be separated by column chromatography to give the desired product 4-substituted-6-amino-7-hydroxy-quinoline-3-carbonitriles 27 where only the 7-alkoxy of 4-substituted-7-alkoxy-6-amino-quinoline-3-carbonitriles 9 is dealkylated. Condensation of 4-substituted-6-amino-7-hydmoxy-quinoline-3-carbonitiiles 27 with dibromides 28 where R4, Z, n, X, G1, G2, G3, and G4 are hereinbefore defined in alcohol solvents including 2-ethoxyethanol, in the presence of potassium carbonate gives the tricyclic compounds 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 of this invention. 
By using similar methods, as shown in Flowsheet 5a, the intermediates 4-substituted-6-alkoxy-7-amino-quinoline-3-carbonitriles 26 may be converted to 3,4-dihydro-2H-[1,4]oxazino[2,3-g]qulinoline-8-carbonitniles of this invention 31. 
The preparation of the tricyclic compounds of this invention encompassed by 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of Formula 34 is described below in Flowsheet 6 wherein Z, n, X, G1, G2, G3, and G4 are hereinbefore defined. R10 is alkyl of 1 to 6 carbon atoms (preferably isobutyl) and R2xe2x80x94 is a radical selected from the group consisting of: 
wherein R6, R3, R5, J, s, r, u, and v are hereinbefore defined.
According to the reactions outlined in Flowsheet 6, acylation of 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 with either acid chlorides of Formula 32 or mixed anhydrides of Formula 33 (which are prepared from the corresponding carboxylic acids) in an ineil solvent such as tetrahydrofuran (THF) in the presence of an organic base selected from pyridine, triethylamine [(C2H5)3N], N, N-diisopr-opylethylamine, and N-methylmorpholine and the like gives 1-substituted-2,3-dihydro-1H-[1,4]oxazinof3,2-g]quinoline-8-carbonitriles 34. In those cases where the acid chlorides 32 or the mixed anhydrides 33 have an asymmetric carbon atom, they may be used as the racemate or as the individual R or S entantiomers wherein the compounds of this invention will be in the racemic or R and S optically active forms, respectively. In those cases, where R2 contains primary or secondary amino groups, the amino groups will first have to be protected prioIr to anhydride or acid chloride formation. Suitable protecting groups include, but are not limited to, teii-butoxycarbonyl (BOC) and benzyloxycarbonyl (CBZ) protecting groups and the like. The BOC protecting group may be removed from the final products by treatment with an acid such as trifluoroactic acid (TFA) while the CBZ protecting group may be removed by catalytic hydrogenation. In those cases where R2 contains hydroxyl groups, the hydroxyl groups may optionally need protection puior to treatment with an anhydride or acid chloride. Suitable protecting groups include, but are not limited to, t-butyldimethylsilyl, tetrahydropyranyl, or benzyl protecting groups. t-Butyldimethylsilyl and tetrahiydropyranyl protecting groups may be removed from the final products by treatment with an acid such as acetic acid or hydrochloric acid while the benzyl protecting group may be removed by catalytic hydrogenation. In those cases, in intermediates 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 where X contains primary or secondary amino groups or-hydroxyl groups, it may be necessary to protect these groups prior to the reaction with acid chlorides 32 or mixed anhydrides 33. The same amine or alcohol protecting groups describe above may be used and they may be removed from the products as previously described. 
In a similar manner, 3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles of Formula 31 may be converted to 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles of Formula 35 as shown in Flowsheet 6a. In those cases, in intermediates 3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles 31 where X contains primary or secondary amino groups or hydroxyl groups, it may be necessary to protect these groups prior to the reaction with acid chlorides 32 or mixed anhydrides 33. The same amine or alcohol protecting groups described above may be used and they may be removed from the products as previously described. 
In order to prepare some of the compounds of this invention certain amines and mixed anhydrides are required; these are prepared as outlined below in the following Flowsheet 7 wherein R3a, R6, R10, Jxe2x80x2, and s are as hereinbefore defined with the proviso that R3aH is an amine. Reaction of amines 170, R3aH with substitued-alkynes 36 is accomplished by heating in an inert solvent such as tetrahydrofuran (THF) or N,N-dimethylfoimamide (DMF), or using potassium or cesium carbonate in acetone to yield aminoalkynes 37. The temperature and duration of the heating will depend on the reactivity of alkynes 36; longer reaction times and higher temperatures may be required when s is greater than 1. Some representative R3axe2x80x94 moieties of amines 170, R3aH are shown below in List A wherein R6, p, and r are as defined above. The amines 170, R3aH are available commercially, are known in the chemical literature, or may be prepared by simple procedures that are well known in the art. In some cases, amines 170, R3aH may have one or more asymmetric carbon atoms; and they may be used as the racemate or they may be resolved and used as the individual R or S entantiomers in which case the compounds of this invention will be in the racemic or optically active forms, respectively. Treatment of aminoalkynes 37 with an alkyl lithium reagent such as n-butyl lithium (n-BuLi) followed by quenching with an atmosphere of dry carbon dioxide (CO2) furnishes alkynoic acids of formula 38. These may be converted to mixed anhydrides of Formula 40 using a reagent such as isobutylchloroformate in an inert solvent such as tetrahydrofuran in the presence of a base such as N-methylmoipholine. These anhydrides may then be used to prepare the compounds of this invention as described in the above flowsheets. 

In order to prepare some of the compounds of this invention certain alcohols, and mixed anhydrides are required; these are prepared as outlined below in Flowsheet 7a wherein R3a, R6, R10, Jxe2x80x2, and s are as defined above with alcohols 171, R3aH . Reaction of alcohols 171, R3aH with alkynes 36 is accomplished using sodium hydride in an inert solvent such as tetrahydrofuran (THF), or other non-nucleophic base such as potassium or cesium carbonate in an inert solvent such as tetrahydrofuran, acetone, or N,N-dimethylformamide to yield alkoxyalkynes 41. In some cases, the alcohol R3aH may also be the solvent of the reaction. Some representative R3axe2x80x94 moieties of alcohols 171, R3aH are shown below in List B wherein R6, p, and r are as defined above. The alcohols 171, R3aH are available commercially, are known in the chemical literature, or may be prepared by simple procedures that are well known in the art. In some cases, these alcohols may have one or more asymmetric carbon atoms; they may be used as the Eracemate or they may be resolved and used as the individual R or S entantiomers in which case the compounds of this invention will be in the racemic or optically active forms, respectively. Treatment of alkoxyalkynes 41 with an alkyl lithium reagent such as n-butyl lithium (n-BuLi) followed by quenching with an atmosphere of dry carbon dioxide furnishes the carboxylic acids of formula 42. These may be converted to mixed anhydrides of formula 43 using a reagent such as isobutylchloroformate in an inert solvent such as tetrahydroftiran in the presence of a base such as N-methylmoipholine. These anhydrides may then be used to prepare the compounds of this invention as described in the above flowsheets. 

As outlined in Flowsheet 8 below wherein G1, G2, G3, G4, R6, R10, X, Z, n, and s are as defined above, alcohols 44 may be protected with, for example, a t-butyl dimethysilyl protecting group by reaction with the respective silyl chloride in methylene chloride (CH2Cl2) in the presence of triethylamine and 4-N,N-dimethylamino pyridine (DMAP). The resulting protected alcohols, 45, may be converted to the acetylenic Grignard reagents which, in turn, are maintained under an atmosphere of dry carbon dioxide to give the carboxylic acids 46. As described above the carboxylic acids 46 may be converted to the mixed anhydrides 48 which on reaction with the 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitiiles 29 gives the corresponding amides 50. In the final step of the sequence, the silyl protecting group is removed by treating with acid in a protic solvent mixture to give 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles represented by Formula 51. 
In the same manner as flowvsheet 8 the corresponding 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonities 52 may be prepared as shown in Flowsheet 8a. 
Compounds of this invention are also prepared as shown below in Flowsheet 9 wherein R3a, G1, G2, G3, G4, R6, R10, Jxe2x80x2, X, Z, n, and s are as defined above with the proviso that R3aH is an amine 170 or an alcohol 171. Treatment of substituted alkynes 53 with an alkyl lithium reagent at low temperature followed by quenching with an atmosphere of dry carbon dioxide fumnishes the carboxylic acids of formula 54. These may be converted to mixed anhydrides of Formula 55 using a reagent such as isobutylchloroformate in an inert solvent such as tetrahydrofuran (THF) in the presence of a base such as N-methylmorpholine. These anhydrides may then be used to prepare the compounds of this invention by the reaction with 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of Formula 29 described in the Flowsheets above. The reaction of the alcohols 171, R3aH and amides 57 is accomplished using sodium hydride or other non-nucleophic base in an inert solvent such as tetiahydrofuran or N,N-dimethylformamide to give 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonittiles of this invention represented by ethers 58. In some cases, the alcohol 171, R3aH may also be the solvent of the reaction. Representative R3axe2x80x94 moieties of alcohols are shown in List B. The reaction of amides 57 with an amine 170, R3aH gives 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of this invention represented by amines 59 is accomplished by heating in an inert solvent such as tetrahydrofuran or N,N-dimethylfoiamide, or using potassium or cesium carbonate in acetone. The temperature and duration of the heating will depend on the reactivity of amides 57; longer reaction times and higher temperatures may be required when s is greater than 1. 
Using methods similar to that summarized above in Flowsheet 9 the corresponding 4-substituted-3,4-dihydro-2Hl-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles represented by ethers 60 and amines 61 may be prepared as shown in Flowsheet 9a. 
Other carboxylic acid chlorides and anhydrides needed to prepare some of the compounds of this invention may be prepared as shown below in Flowsheet 10, 10a, and 10b wherein R3a, R6, R3, R10, Z, Qxe2x80x2, Jxe2x80x2, and s are as defined above. The esters 62, 66, or 71 may be hydrolyzed with a base such as barium hydroxide [Ba(OH)2] to give the respective carboxylic acid 63, 67, or 72. These acids may be converted to the respective carboxylic acid chlorides 64 or 69 by using oxalyl chloride and catalytic N,N-dimethylformamide in an inert solvent or respective mixed anhydrides 68 or 73 by using isobutyl chloroformate and an organic base such as N-methylmorpholine. The leaving group Jxe2x80x2 in esters represented by Formula 65 and 70 may be displaced by the amines 170, R3aH or the alcohols 171, R3aH by using procedures previously described to give intermediate ethers 66 and amines 71, respectively. Representative R3axe2x80x94 moieties of amines 170 and alcohols 171 are shown in List A and List B above wherein R6, p, and r are as defined above. The carboxylic acid chlorides 64 and 69 and the anhydrides 68 and 73 may be used to prepare some of the compounds of this invention by using the methods outlined herein above in the Flowsheets. 
By using the methods identical to those outlined above in Flowsheet 10, 10a and 10b, it is possible to prepare the analogous carboxylic acid chlorides and anhydrides given below in List C wherein R3a, R6, R3, p, and s are as previously defined with the proviso that R3a is Jxe2x80x2, an amine 170 or an alcohol 171 where R10 is hereinbefore defined and G is the radical: 
By making use of these carboxylic acid chlorides and anhydrides, by following the methods summarized in the above Flowsheets, compounds of this invention may be prepared. 
Tricyclic compounds of this invention represented by Formulas 77-78 may be prepared as shown in Flowsheet 11 wherein R3a, G1, G2, G3, G4, R6, X, Z, Jxe2x80x2, n, and s are as defined above. The reaction of the carboxylic acid chlorides 74 and 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 using an organic base in an inert solvent gives 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of this invention represented by Formula 76. The reaction of the tricyclic compounds 76 with an alcohol 171, R3aH is accomplished using sodium hydride or other non-nucleophic base such as potassium or cesium carbonate in an inert solvent such as tetrahydrofuran, acetone, or N,N-dimethylformamide to give 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitrile of this invention represented by ethers 77. In some cases, the alcohol 171 R3aH may also be the solvent of the reaction. The reaction of tricyclic compounds 76 with an amine 170, R3aH to give 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitrile of this invention represented by amines 78 is accomplished by heating in an inert solvent such as tetrahydrofuran or N,N-dimethylformamide. Some representative R3axe2x80x94 moieties of amines are shown in List A above and some representative R3axe2x80x94 moieties of alcohols are shown in List B. The temperature and duration of the heating will depend on the reactivity of the tricyclic compounds 76; longer reaction times and higher temperatures may be required when s is greater than 1. In addition, by using this method, the carboxylic acid chlorides and mixed anhydrides listed in List C may be used to prepare the analogous compounds of this invention. 
By applying the methods summarized above, the corresponding 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles represented by ethers 79 and amines 80 may be prepared as shown in Flowsheet 11a. 
The reaction of 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 76 with nitrogen containing heterocycles represented by Formula 81 which also contains an unsaturated carbon-nitrogen bond is accomplished by refluxing in an inert solvent and gives the 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 82 of this invention where the compounds bear a positive charge as shown in Flowsheet 11b. The counter anion Jxe2x80x2 may be replaced with any other pharmaceutically acceptable anion using the appropriate ion exchange resin. The corresponding 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles 82a may be prepared in an analogous manner. 
Some of the compounds of this invention may be prepared as outline below in Flowsheet 12 wherein G1, G2, G3, G4, R6, R10, X, Z, Jxe2x80x2, p, n, and s are as defined above. The substituted acetylenic alcohols 83 may be coupled to the halides, mesylates, or tosylates 84 using a base such as sodium hydride in an inert solvent such as tetrahydrofuran. The resulting acetylene, 85, may then be treated with an alkyl lithium reagent at low temperature. Maintaining the reaction under an atmosphere of carbon dioxide then gives the carboxylic acids 86. These, in turn, are reacted with the 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29, via the mixed anhydrides to give the tricyclic amide of this invention represented by Formula 88. Alternatively, the acetylene intermediates 85 may be prepared starting with an alcohol 89 by first treating with a base such as sodium hydride in an inert solvent such as tetrahydrofuran and then adding an acetylene 90 having an appropriate leaving group Jxe2x80x2 wherein Jxe2x80x2, p and s are hereinbefore defined. 
In a similar manner, as shown in Flowsheet 12, the amino alcohols represented by the formula 89a: (R6)2Nxe2x80x94(C(R6)2)pxe2x80x94OH may be reacted with acetylenes 90 and subsequently converted to the 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of this invention represented by the formula 88a as shown in Flowsheet 12a. 
In an entirely analogous manner to Flowsheet 12, the corresponding 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles 92 are prepared as shown in Flowsheet 12b. 
In a similar manner, to Flowsheet 12a, the amino alcohols represented by the formula 91: (R6)2Nxe2x80x94(C(R6)2)pxe2x80x94OH may be reacted with acetylene 90 and subsequently converted to the 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles of this invention represented by the formula 92a as shown in Flowsheet 12c. 
Disulfide compounds of this invention represented by Formula 98 may be prepared as shown in Flowsheet 13 wherein G1, G2, G3, G4, R6, R3, R10, X, Z, n, and r are as defined above. The reaction of the mecapto carboxylic acids 93 with the disulfide reagents 94 give the disulfide compounds represented by Formula 95. Alternatively, disulfides 95 may be prepared from the mercaptan R3SH using the mercapto acid 93, triethylamine and 2,2xe2x80x2-dipyridyl disulfide. Mixed anhydride formation to give mixed anhydrides 96 followed by condensation with the 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 give amides of this invention represented by Formula 98. In those cases, where R3 contains primary or secondary amino groups, the amino groups will first have to be protected prior to anhydride or acid chloride formation. Suitable protecting groups include, but are not limited to, tert-butoxycarbonyl (BOC) and benzyloxycarbonyl (CBZ) protecting groups. The BOC protecting group may be removed from the final products by treatment with an acid such as trifluoroactic acid (TFA) while the CBZ protecting group may be removed by catalytic hydrogenation. 
In an entirely analogous manner to Flowsheet 13, the corresponding 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles 98a are prepared as shown in Flowsheet 13b. In those cases where R3 contains hydroxyl groups, the hydroxyl groups may have to be protected prior to treatment with an anhydride or acid chloride. Suitable protecting groups include, but are not limited to, t-butyldimethylsilyl, tetrahydropyranyl, or benzyl protecting groups. t-Butyldimethylsilyl and tetrahydropyranyl protecting groups may be removed from the final products by treatment with an acid such as acetic acid or hydrochloric acid while the benzyl protecting group may be removed by catalytic hydrogenation. 
Compounds of this invention represented by Formulas 101-103 may be prepared as shown in Flowsheet 14 wherein Q, G1, G2, G3, G4, R5, Jxe2x80x2, X, Z, and n are as defined above. Alkylation of 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 with formula 99 may be accomplished by heating in an inert solvent such as N,N-dimethylformamide using a base such as potassium carbonate to give 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of this invention represented by the Formula 101. When Q is alkoxy, the ester group may be hydrolyzed to an acid using a base such as sodium hydroxide in methanol. In a similar manner, by using intermediates 104 and 105, 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of this invention represented by Formulas 102 and 103 respectively, may be prepared. 
In an entirely analogous manner to Flowsheet 14, the corresponding 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles 101a, 102a, and 103a are prepared as shown in Flowsheet 14a. 
Compounds of this invention represented by Formula 108 may be prepared as shown in Flowsheet 15 wherein G1, G2, G3, G4, R5, X, Z, and n are as defined above. The reaction of reagent 106 with the 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 is accomplished by using an excess of an organic base such as triethylamine and an inert solvent such as tetrahydrofuran to give sulfonamides of this invention represented by Formula 108. 
In an entirely analogous manner to Flowsheet 15, the sulfonamides represented by Formula 108a are prepared as shown in Flowsheet 15a. 
Compounds of this invention represented by Formula 111 may be prepared as shown in Flowsheet 16 wherein G1, G2, G3, G4, R11, X, Z, and n are as defined above. The reaction of 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 and aldehydes 109 using sodium borohydride (NaBH4) in N,N-dimethylformamide and trifluoroacetic acid at room temperature gives 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of this invention represented by 111. 
In the same manner, to Flowsheet 16, the 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles represented by Formula 112 may be prepared as shown in Flowsheet 16a. 
Compounds of this invention represented by Formula 116 and 117 may be prepared as shown in Flowsheet 17 wherein R13, G1, G2, G3, G4, R6, Jxe2x80x2, X, Z, k and n are as defined above. The reaction of 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 and aldehydes 113 using sodium borohydride in dimethylformamide and trifluoroacetic acid at room temperature gives the compounds of this invention represented by 115 containing a leaving group Jxe2x80x2. Displacement of Jxe2x80x2 in 115 with an alcohol 173, R13H where some representative R13xe2x80x94 moieties of alcohols 173, R13H are shown in List E below, is accomplished by using sodium hydride or other non-nucleophic base in an inert solvent such as tetrahydrofuran or N,N-dimethylformamide to give ethers of this invention represented by Formula 116. In some cases, the alcohol 173, R13H may also be the solvent of the reaction. Displacement of the leaving group Jxe2x80x2 in 115 with an amine 172, R13H of List D is accomplished by heating in an inert solvent such as tetrahydrofuran or N,N-dimethylformamide, or using potassium or Cesium carbonate in acetone to give amines of this invention represented by 117.


In the same manner, as shown in Flowsheet 17, the corresponding 3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles represented by ethers 118 and amines 119 may be prepared as shown in Flowsheet 17a. 
Compounds of this invention represented by Formula 122 may be prepared as shown in Flowsheet 18 wherein R12, G1, G2, G3, G4, Jxe2x80x2, X, Z, and n are as defined above. Treatment of 2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles 29 with Formula 120 containing a leaving group Jxe2x80x2 in an inert solvent such as tetrahydrofuran gives 1-substituted-2,3-dihydro-1H-[1,4]oxazino[3,2-g]quinoline-8-carbonitriles of this invention represented by Formula 122. 
Using methods similar to that summarized above in Flowsheet 18, 4-substituted-3,4-dihydro-2H-[1,4]oxazino[2,3-g]quinoline-8-carbonitriles represented by Formula 123 may be prepared. 
The preparation of compounds and intermediates of this invention, 4-substituted-quinoline-3-carbonitriles 130 is described below in Flowsheet 19 where X, n, and Z are as hereinbefore described. Substituted-anilines 124 which may be prepared from 3-aminothiophenol is condensed with 2-cyano-3-ethoxy-acrylic acid ethyl ester 3 by heating in the absence of solvent to give esters 125, wherein R4a is a thioprotecting group which is selected from benzyl, t-butyl and the like. Thermal cyclization of esters 125 in refluxing 3:1 diphenyl ether/biphenyl or diphenyl ether gives 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 126, which may also exist in the 4-hydroxy-quinoline tautomeric form. Nitration of 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 126 in trifluoroacetic acid (TFA) with ammonium nitrate at room temperature gives the nitro compounds 127. Nitro compounds 127 are refluxed with chlorinating reagent selected from the group consisting of phosphorous oxychloride, oxalyl chloride or phosphorous oxychloride and phosphorus pentachloride to furnish 4-chloro compounds 128. Condensation of 4-chloro compounds 7 with various amines, anilines, alcohols, phenols, mercaptans, and thiophenols of the Formula HZxe2x80x94(CH2)nxe2x80x94X 7a where Z, X and n are hereinbefore defined gives 4-substituted-6-nitro-quinoline-3-carbonitriles 129. This condensation may be accelerated by heating the reaction mixture together with one equivalent of pyridine hydrochloride in alcohol solvents which include isopropanol and 2-ethoxyethanol or by using bases such as trialkylamines, sodium hydride in an inert solvent which includes tetrahydrofuran (THF), sodium or potassium alkoxides in alcohol solvents which includes ethanol, and the like. Reduction of 4-substituted-6-nitro-quinoline-3-carbonitriles 129 with iron and ammonium chloride in refluxing methanol and water furnishes 4-substituted-6-amino-quinoline-3-carbonitriles 130. 
The prepartion of compounds of Formula 130, which are important intermediates for the preparation of compounds of this invention, are prepared by an alternate route as described below in Flowsheet 20 where R4a, Z, n and X are hereinbefore defined. Nitroanilines 131 which may be prepared from 2-amino-5-nitrothiophenol, are acetylated with acetic anhydride (Ac2O) and water at room temperature to give nitro compounds 132. Reduction of nitro compounds 132 with iron and ammonium chloride in refluxing methanol and water furnishes anilines 133. The condensation of anilines 133 and 2-cyano-3-ethoxy-acrylic acid ethyl ester 3 by heating in the absence of solvent gives esters 134. Thermal cyclization of esters 134 in refluxing 3:1 diphenyl ether/biphenyl or diphenyl ether gives 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 135. Chlorination of 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 135 in refluxing chlorinating reagent selected from phosphorous oxychloride and oxalyl chloride furnishes 4-chloro-quinolines 136. Condensation of 4-chloro-quinolines 136 with various amines, anilines, alcohols, phenols, mercaptans, and thiophenols of the Formula HZxe2x80x94(CH2)nxe2x80x94X 7a where Z, X and n are hereinbefore defined gives 4-substituted-6-acetamido-quinoline-3-carbonitriles 137. The condensation may be accelerated by heating the reaction mixture together with one equivalent of pyridine hydrochloride in alcohol solvents which include isopropanol and 2-ethoxyethanol or by using bases such as trialkylamines, sodium hydride in an inert solvent which includes tetrahydrofuran (THF), sodium or potassium alkoxides in alcohol solvents which includes ethanol, and the like. Hydrolysis of 4-substituted-6-acetamido-quinoline-3-carbonitriles 137 in aqueous hydrochloric acid or sodium hydroxide in methanol gives 4-substituted-6-amino-quinoline-3-carbonitriles 130. 
The preparation of the compounds of this invention encompassed by compounds of Formula 145, which are important intermediates for the preparation of compounds of this invention, is described below in Flowsheet 21 where R4a, Z, n and X are hereinbefore defined. Nitroanilines 138 which may be prepared from 2-amino-4-nitrothiophenol, are acetylated with acetic anhydride (Ac2O) and water at room temperature to give nitro compounds 139. Reduction of nitro compounds 139 with iron and ammonium chloride in refluxing methanol and water furnishes anilines 140. The condensation of anilines 140 and 2-cyano-3-ethoxy-acrylic acid ethyl ester 3 by heating in the absence of solvent gives esters 141. Thermal cyclization of esters 141 in refluxing 3:1 diphenyl ether/biphenyl or diphenyl ether gives 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 142. Chlorination of 4-oxo-1,4-dihydro-quinoline-3-carbonitriles 142 in refluxing chlorinating reagent selected from phosphorous oxychloride and oxalyl chloride gives 4-chloro-quinolines 143. Condensation of 4-chloro-quinolines 143 with various amines, anilines, alcohols, phenols, mercaptans, and thiophenols of Formula HZxe2x80x94(CH2)nxe2x80x94X 7a where Z, X and n are hereinbefore defined gives the compounds 4-substituted-quinoline-3-9 carbonitriles 144. This condensation may be accelerated by heating the reaction mixture together with one equivalent of pyridine hydrochloride in alcohol solvents which include isopropanol and 2-ethoxyethanol or by using bases which includes trialkylamines, sodium hydride in an inert solvent which includes tetrahydrofuran (THF), and the like, sodium or potassium alkoxides in alcohol solvents which includes ethanol, and the like. Hydrolysis of 4-substituted-7-acetamido-quinoline-3-carbonitriles 144 in aqueous hydrochloric acid or sodium hyroxide in methanol gives 4-substituted-7-amino-quinoline-3-carbonitriles 145. 
The preparation of the compounds and intermediates of this invention encompassed by Formula 147 is described below in Flowsheet 22 where R4a, Z, n, X, G1, G2, G3, and G4 are hereinbefore defined. Deprotecting the thiol group of 4-substituted-6-amino-quinoline-3-carbonitriles 130 may be accomplished by deprotecting procedures such as, but not limited to, HF in anisole or with mercuric trifluoroacetate, trifluoroacetic acid and anisole to give aminothiophenols 146. Condensation of aminothiophenol 146 with dibromides 28 where Z, n, X, G1, G2, G3, and G4 are hereinbefore defined in alcohol solvents including 2-ethoxyethanol, in the presence of potassium carbonate gives the tricyclic compounds 2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles 147 of this invention. 
By using similar methods, the intermediates thio-protected anilines 145 may be converted to 3,4-dihydro-2H-[1,4]thiazino[2,3-g]quinoline-8-carbonitriles of this invention 149 as shown in Flowsheet 23. 
According to the reactions outlined in Flowsheet 24, acylation of 2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles 147 with either acid chlorides of Formula 32 or mixed anhydrides of Formula 33 (which are prepared from the corresponding carboxylic acids) in an inert solvent such as tetrahydrofuran (THF) in the presence of an organic base selected from pyridine, triethylamine [(C2H5)3N], N,N-diisopropylethylamine, and N-methylmorpholine gives 1-substituted-2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles 150. In those cases where the acid chlorides 32 or the mixed anhydrides 33 have an asymmetric carbon atom, they may be used as the racemate or as the individual R or S entantiomers wherein the compounds of this invention will be in the racemic or R and S optically active forms, respectively. In those cases, where R2 contains primary or secondary amino groups, the amino groups will first have to be protected prior to anhydride or acid chloride formation. Suitable protecting groups include, but are not limited to, tert-butoxycarbonyl (BOC) and benzyloxycarbonyl (CBZ) protecting groups. The BOC protecting group may be removed from the final products by treatment with an acid such as trifluoroactic acid (TFA) while the CBZ protecting group may be removed by catalytic hydrogenation. In those cases where R2 contains hydroxyl groups, the hydroxyl groups may first have to be protected prior to treatment with an anhydride or acid chloride. Suitable protecting groups include, but are not limited to, t-butyldimethylsilyl, tetrahydropyranyl, or benzyl protecting groups. t-Butyldimethylsilyl and tetrahydropyranyl protecting groups may be removed from the final products by treatment with an acid such as acetic acid or hydrochloric acid while the benzyl protecting group may be removed by catalytic hydrogenation. In those cases, in intermediates 2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles 150 where X contains primary or secondary amino groups or hydroxyl groups, it may be necessary to protect these groups prior to the reaction with acid chlorides 32 or mixed anhydrides 33. The same amine or alcohol protecting groups describe above may be used and they may be removed from the products as previously described. 
In a similar manner, 3,4-dihydro-2H-[1,4]thiazino[2,3-g]quinoline-8-carbonitriles of Formula 149 may be converted to 4-substituted-3,4-dihydro-21H-[1,4]thiazino[2,3-g]quinoline-8-carbonitriles of Formula 151 as shown in Flowsheet 25. In those cases, in intermediates 3,4-dihydro-2H-[1,4]thiazino[2,3-g]quinoline-8-carbonitriles 149 where X contains primary or secondary amino groups or hydroxyl groups, it may be necessary to protect these groups prior to the reaction with acid chlorides 32 or mixed anhydrides 33. The same amine or alcohol protecting groups described above may be used and they may be removed from the products as previously described. 
Compounds of this invention represented by Formulas 152-154 may be prepared as shown in Flowsheet 26 wherein Q, G1, G2, G3, G4, R5, Jxe2x80x2, X, Z, and n are as defined above. Akylation of 2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles 147 with 99 may be accomplished by heating in an inert solvent such as N,N-dimethylformamide using a base such as potassium carbonate to give 1-substituted-2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles of this invention represented by the Formula 152. When Q is alkoxy, the ester group may be hydrolyzed to an acid using a base such as sodium hydroxide in methanol. In a similar manner, by using intermediates 104 and 105, 1-substituted-2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles of this invention represented by Formulas 153 and 154 respectively, may be prepared. 
In an entirely analogous manner to Flowsheet 26, the corresponding 4-substituted-3,4-dihydro-2H-[1,4]thiazino[2,3-g]quinoline-8-carbonitriles 155, 156, and 157 are prepared as shown in Flowsheet 27. 
Compounds of this invention represented by Formula 158 may be prepared as shown in Flowsheet 28 wherein G1, G2, G3, G4, R5, X, Z, and n are as defined above. The reaction of reagent 106 with the 2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles 147 is accomplished by using an excess of an organic base such as triethylamine and an inert solvent such as tetrahydrofuran to give sulfonamides of this invention represented by Formula 158. 
In an entirely analogous manner as shown in Flowsheet 28, the sulfonamides represented by Formula 159 are prepared as shown in Flowsheet 29. 
Compounds of this invention represented by Formula 160 may be prepared as shown in Flowsheet 30 wherein G1, G2, G3, G4, R11, X, Z, and n are as defined above. The reaction of 2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles 147 and aldehydes 109 using sodium borohydride (NaBH4) in N,N-dimethylformamide and trifluoroacetic acid at room temperature gives 1-substituted-2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles of this invention represented by 160. 
In the same manner, as shown in Flowsheet 30, the 4-substituted-3,4-dihydro-2H-[1,4]thiazino[2,3-g]quinoline-8-carbonitriles represented by Formula 161 may be prepared as shown in Flowsheet 31. 
Compounds of this invention represented by Formula 162 may be prepared as shown in Flowsheet 32 wherein R12, G1, G2, G3, G4, Jxe2x80x2, X, Z, and n are as defined above. Treatment of 2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles 147 with Formula 120 containing a leaving group Jxe2x80x2 in an inert solvent such as tetrahydrofuran gives 1-substituted-2,3-dihydro-1H-[1,4]thiazino[3,2-g]quinoline-8-carbonitriles of this invention represented by Formula 162. 
Using methods similar to that summarized above in Flowsheet 32, 4-substituted-3,4-dihydro-2H-[1,4]thiazino[2,3-g]quinoline-8-carbonitriles represented by Formula 163 may be prepared. 
With respect to the above Flowsheets 1-33, in those cases where R1, or other substituent may contain an asymmetric carbon atom, the intermediates may be used as the racemate or as the individual R or S entantiomers in which case the compounds of this invention will be in the racemic or R and S optically active forms, respectively. In cases where the substituents may contribute more than one asymmetric carbon atom, diasteriomers may be present; these may be separated by methods well known in the art including, but not limited to, fractional crystallization and chromatographic methods. In those cases where R1 or other substituents contain primary or secondary amino groups, the amino groups may first have to be used in protected form prior to applying the chemistry described in the above Flowsheets. Suitable protecting groups include, but are not limited to, tert-butoxycarbonyl (BOC) and benzyloxycarbonyl (CBZ) protecting groups. The former protecting group may be removed from the final products by treatment with an acid such as trifluoroactic acid while the latter protecting group may be removed by catalytic hydrogenation. In those cases where R1 or other substituents contain hydroxyl groups, the hydroxyl groups may first have to be used in protected form prior to applying the chemistry described in the above Flowsheets. Suitable protecting groups include, but are not limited to, t-butyldimethylsilyl, tetrahydropyranyl, or benzyl protecting groups. The first two protecting groups may be removed from the final products by treatment with an acid such as acetic acid, hydrofluoric acid, or hydrochloric acid while the latter protecting group may be removed by catalytic hydrogenation.
Representative compounds of this invention were evaluated in several standard pharmacological test procedures that showed that the compounds of this invention possess significant activity as inhibitors of protein kinases and are antiproliferative agents. Disease states which can be treated or inhibited by protein kinase inhibitors include those in which the etiology is at least in part caused by a defect upstream in a signaling pathway from a protein kinase (i.e., colon cancer); those in which the etiology is at least in pail caused by an overexpressed protein kinase (i.e., lung cancer and colonic polyps); and those in which the etiology is at least in part caused by a dysregulated protein kinase (gene turned on at all times; glioblastoma).
Based on the activity shown in the standard pharmacological test procedures, the compounds of this invention are therefore useful as antineoplastic agents. In particular, these compounds are useful in treating, inhibiting the growth of, or eradicating neoplasms such as those of the breast, kidney, bladder, mouth, larynx, esophagus, stomach, colon, ovary, lung, pancreas, liver, prostate and skin.
In addition to having antineoplastic properties, the compounds of the present invention are useful in treating or inhibiting a variety of protein tyrosine kinase-associated disorders including: polycystic kidney disease, colonic polyps, restenosis; atherosclerosis; angiofibromas; hemangiomas; diabetes; acute and chronic nephropathies; Kaposi""s sarcoma; neovascularization associated with macular degeneration; rheumatoid arthritis; osteoarthritis; transplant rejection; psoriasis; lupus; graft versus host disease; glomerulonephritis; respiratory and skin allergies; autoimmune alopecia; Autoimmune Hyperthyroidism; multiple sclerosis; atopic dermatitis; and systemic sclerosis; and are useful as antibacterial and antiviral agents.
As used in accordance with this invention, the term providing an effective amount of a compound means either directly administering such compound, or administering a prodrug, derivative, or analog which will form an effective amount of the compound within the body.
In addition to the above utilities some of the compounds of this invention are useful for the preparation of other compounds of this invention.
The test procedures used and results obtained are shown below.
Representative compounds of this invention were evaluated in several standard pharmacological test procedures that showed that the compounds of this invention possess significant activity as inhibitors of protein tyrosine kinase and are antiproliferative agents. Based on the activity shown in the standard pharmacological test procedures, the compounds of this invention are therefore useful as antineoplastic agents. The test procedures used and results obtained are shown below.
Representative test compounds were evaluated for their ability to inhibit the phosphorylation of the tyrosine residue of a peptide substrate catalyzed by the enzyme epidermal growth factor receptor kinase. The peptide substrate (RR-SRC) has the sequence arg-arg-leu-ile-glu-asp-ala-glu-tyr-ala-ala-arg-gly. The enzyme used in this assay is the His-tagged cytoplasmic domain of EGFR. A recombinant baculovirus (vHcEGFR52) was constructed containing the EGFR cDNA encoding amino acids 645-1186 preceded by Met-Ala-(Ris)6. Sf9 cells in 100 mm plates were infected at an moi of 10 pfu/cell and cells were harvested 48 hours post infection. A cytoplasmic extract was prepared using 1% Triton X-100 and applied to Ni-NTA column. After washing the column with 20 mM imidazole, HcEGFR was eluted with 250 mM imidazole (in 50 mM Na2RPO4, pH 8.0, 300 mM NaCl). Fractions collected were dialyzed against 10 mM HEPES, pH 7.0, 50 mM NaCl, 10% glycerol, 1 xcexcg/mL antipain and leupeptin and 0.1 mM Pefabloc SC. The protein was frozen in dry ice/methanol and stored xe2x88x9270xc2x0 C.
Test compounds were made into 10 mg/mL stock solutions in 100% dimethylsulfoxide (DMSO). Prior to experiment, stock solutions were diluted to 500 xcexcM with 100% DMSO and then serially diluted to the desired concentration with HEPES buffer (30 mM HEPES pH 7.4).
For the enzyme reaction, 10 xcexcL of each inhibitor (at various concentrations) were added to each well of a 96-well plate. To this was added 3 xcexcL of enzyme (1:10 dilution in 10 mM HEPES, pH 7.4 for final conc. of 1:120). This was allowed to sit for 10 minutes on ice and was followed by the addition of 5 xcexcl peptide (80 uM final conc.), 10 xcexcl of 4xc3x97Buffer (Table A), 0.25 xcexcL 33P-ATP and 12 xcexcL H2O. The reaction was allowed to run for 90 minutes at room temperature and was followed by spotting the entire volume on to precut P81 filter papers. The filter discs were washed 2xc3x97 with 0.5% phosphoric acid and radioactivity was measured using a liquid scintillation counter.
The inhibition data for representative compounds of the invention are shown below in TABLE 1. The IC50 is the concentration of test compound needed to reduce the total amount of phosphorylated substrate by 50%. The % inhibition of the test compound was determined for at least three different concentrations and the IC50 value was evaluated from the dose response curve. The % inhibition was evaluated with the following formula:
xe2x80x83% inhibition=100xe2x88x92[CPM(drug)/CPM(control)]xc3x97100
where CPM(drug) is in units of counts per minute and is a number expressing the amount of radiolabeled ATP (xcex3-33P) incorporated onto the RR-SRC peptide substrate by the enzyme after 90 minutes at room temperature in the presence of test compound as measured by liquid scintillation counting. CPM(control) is in units of counts per minute and was a number expressing the amount of radiolabeled ATP (xcex3-33P) incorporated into the RR-SRC peptide substrate by the enzyme after 90 minutes at room temperature in the absence of test compound as measured by liquid scintillation counting. The CPM values were corrected for the background counts produced by ATP in the absence of the enzymatic reaction. Where it was possible to determine an IC50 value, this is reported in TABLE 1 otherwise the % inhibition at 0.5 xcexcM concentration of test compound is shown in TABLE 1.
The c-Met kinase assay was performed as a DELFIA (dissociation enchanced lanthanide fluorometric immunoassay), an ELISA-like protocol based upon time-resolved fluorometry as described previously [Braunwalder, A. F., Yarwood, D. R., Sills, M. A., and Lipson, K. E., Anal. Biochem. 238, 159-164 (1996)]. The protocol was adopted for the screening of inhibitors of the kinase activity of recombinant c-Met. [Loganzo, F., and. Hardy, C., American Biotechnology Laboratory 16(13), 26-28 (1998)]. The cytoplasmic domain of c-Met was generated by reverse transcriptase-polymerase chain reaction using RNA isolated from human mammary epithelial cells as template, cloned into the pFastBac-HTc vector (Life Technologies) and protein expressed in Sf9 insect cells by baculovirus infection. Protein was purified by imidazole elution from Ni-NTA resin (Invitrogen, Carlsbad, Calif.) or Talon resin (Clontech, Palo Alto, Calif.). Various conditions were evaluated for the kinase screening protocol and screening was accomplished with the following method. Polystyrene Maxisorp plates (Nunc) were pre-coated for 1 hour to overnight at room temperature with 5 xcexcg/ml poly(Glu4-Tyr) diluted in Tris-buffered saline (TBS). Plates were washed 3xc3x97 with TBS. c-Met protein preparation was diluted 1:80 into 0.1% BSA/4 mM HEPES. A master mix was prepared consisting of: 1 volume of diluted c-Met, 1 volume of 5xc3x97buffer (20 mM HEPES, pH 7.4; 2 mM MnCl2; 100 xcexcM Na3VO4; 1 mM DTT), 0.9 volume of water. Master mix (29 xcexcl) was added to the wells of peptide-coated microtiter plates. For assays where c-Met autophosphorylation was evaluated, plates were uncoated and c-Met was diluted into 4 mM HEPES at 1:40 without BSA. Compounds (1 xcexcl) at various dilutions (1 mM to 0.1 xcexcM) prepared in DMSO were added and mixed well with an automatic piptettor. After incubation 20 minutes at room temperature, the reaction was initiated with 20 xcexcl of 62.5 xcexcM ATP/50 mM MgCl2 (final 25 xcexcM ATP/20 mM MgCl2 in assay). Reactions were incubated 45 minutes at room temperature. Plates were then washed 3xc3x97 with 100 xcexcl each of DELFIA wash buffer (Wallac). Antibody binding was performed by adding 75 xcexcl of Europium-conjugated anti-phosphotyrosine diluted 1:2000 in Assay Buffer (Wallac) for 1 hour at room temperature. Plates were washed 3xc3x97 with DELFIA wash buffer. Detection was done by addition of 100 xcexcl of Enhancement Solution (Wallac). After 30 minutes, plates were read in a Wallac VICTOR-2 time-resolved fluorometer. Inhibition data was reported as europium counts for compound treated wells normalized to control counts. IC50 values were obtained by using log doses of compound and calculated using Data Analysis Toolbox software, version 1.0.2 (MDL Information Systems, Inc.).
Inhibitors of p60c-src (partially purified preparation purchased from Upstate Biotechnologies) tyrosine kinase activity are analyzed in an ELISA format. The Boehringer Mannheim Tyrosine Kinase Assay Kit (Catalog number 1-534505) with a cdc2 substrate peptide containing Tyr15 is used for the assay. HRP-conjugated anti-phosphotyrosine is used to detect phosphorylated peptide via a color reaction. Conditions recommended by the manufacturer are employed.
Reaction conditions: Five microliter aliquots of each compound prepared fresh at the time of the assay are added as a solution in 10 mM HEPES pH 7.5, 10% DMSO to the reaction well. Thirty-five microliters of reaction mix containing Src, buffer and peptide/bovine serum albumin mix are added to the compound wells and incubated at 30xc2x0 C. for 10 minutes (reaction buffer: 50 mM TrisHCl pH 7.5, 10 mM MgCl2, 0.1 mM EGTA, 0.5mM Na3VO4). The reaction is started by addition of 10 microliters of ATP, incubated at 30xc2x0 C. for 1 hour, and stopped by addition of 20 microliters of 0.5M EDTA. The reaction mixture with the phosphorylated peptide was then transferred to a streptavidin-coated microtiter plate (provided in the kit) and allowed to bind for 20 minutes. Unbound peptide and reaction mixture was decanted and the plate was washed with PBS six times. Horseradish peroxidase-conjugated phosphotyiosine antibody supplied in the kit was incubated with the plate for one hour, then decanted. The plate was again washed with PBS six times. Substrate (provided in the kit) was added and absorbance at 405 nm was measured.
Activity was determined as % inhibition as calculated by the formula:
(1xe2x88x92Abs/Abs(max))xc3x97100=% inhibition.
Where multiple concentrations of the test agent were used, an IC50 (concentration which gives 50% inhibition) could be determined.
The result obtained for representative compound of this invention is listed in Table 3.
Raf-1 (c-Raf) is used to phosphorylate and activate inactive GST-MEK1 which then can phosphorylate and activate inactive p42 GST-MAPK, which subsequently is measured for phosphorylation of the TEY sequence (aa""s 202-204) by a phospho-specific antibody from Sigma (cat. #77439219041) Reagents: Sf9 insect cell lysate containing full length 6his-tagged recombinant human c-Raf. (Specific Activity: xcx9c200 U/ml). Human Non-active Mek-1-GST and human GST-MAP kinase (recombinant proteins produced in E. coli).
Stock Solutions c-Raf Assay:
1. Assay Dilution Buffer (ADB): 20 mM MOPS, pH 7.2, 25 mM xcex2-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol.
2. Magnesium/ATP Cocktail: 500 xcexcM cold ATP and 75 mM magnesium chloride in ADB.
4. Active Kinase: Human Active c-Raf: Use at 0.4 U per assay point.
5. Non-active GST-MEK1: Use at 0.1 xcexcg per assay point.
6. Non-active GST-p42 MAP Kinase: Use at 1.0 xcexcg per assay point.
Stock Solutions ELISA:
1. TBST-Tis (50 mM, pH 7.5), NaCl (150 mM), Tween-20 (0.05%)
2. Superblock (Pierce)
3. Anti-GST Ab (Pharmacia)
4. Anti-Phospho MAPK (Sigma)
5. Anti-Mouse Ab/Europium conjugate (Wallac) Assay Procedure:
First Stage: c-Raf Dependent Activation of GST-MEK and GST-MAPK
1. Add 20 ml of ADB per assay (i.e. per well of a 96 well plate)
2. Add 10 ml of 0.5 mM cold ATP and 75 mM magnesium chloride in ADB.
3. Add 2 ml of c-Raf (0.4U/assay), in conjunction with 1.6 ml non-active MEK1 (0.4 mg/assay).
4. Add 4 ml of non-active GST-p42 MAP Kinase (1.0 mg/assay).
5. Incubate for 60 minutes at 30xc2x0 C. in a shaking incubator.
6. Transfer this mixture to an anti-GST Ab coated 96 well plate (Nunc Immunosorb plates coated o/n with a-GST, then blocked with Pierce Superblock).
7. Incubate for 60 minutes at 30xc2x0 C. in a shaking incubator.
8. Wash 3xc3x97 with TBST, add Anti-Phospho MAPK (Sigma) (1:3000).
9. Incubate for 60 minutes at 30xc2x0 C. in a shaking incubator.
10. Wash 3xc3x97 with TBST, add Anti-Mouse Ab/Europium conjugate (Wallac) (1:500).
11. Incubate for 60 minutes at 30xc2x0 C. in a shaking incubator.
12. Wash 3xc3x97 with TBST, Read plates in Wallac Victor model Plate Reader.
13. Collect data analyze in Excel for single point and IC50 determinations.
Single point assayxe2x80x94% inhibition at 10 mg/ml (% Inhibition=1xe2x88x92cpd.treated sample/untreated control). Typically Raf-1 assay is run at compound concentrations from 10 xcexcM to 30 nM in half log dilutions. (% inhibition is determined for each compound concentration). The results obtained for representative compound of this invention are listed in Table 4.
Human tumor cell lines were plated in 96-well plates (250 xcexc/well, 1-6xc3x9710 104 cells/ml) in RPMI 1640 medium, containing 5% FBS (Fetal Bovine Serum). Twenty four hours after plating, test compounds were added at five log concentrations (0.01-100 mg/ml) or at lower concentrations for the more potent compounds. After 48 hours exposure to test compounds, cells were fixed with trichloroacetic acid, and stained with Sulforhodamine B. After washing with trichloroacetic acid, bound dye was solubilized in 10 mM Tris base and optical density was determined using a plate reader. Under conditions of the assay the optical density is proportional to the number of cells in the well. IC50s (concentrations causing 50% inhibition of cell growth) were determined from the growth inhibition plots. The test procedure is described in details by Philip Skehan et. al, J. Natl. Canc. Just., 82, 1107-1112 (1990). These data are shown below in TABLE 5. Information about some of the cell lines used in these test procedures is available from the American Type Tissue Collection: Cell Lines and Hybridomas, 1994 Reference Guide, 8th Edition. The Her2Neu cell line is a 3T3 line that has been transfected with Her2 receptor kinase.
BALB/c nu/nu female mice (Charles River, Wilmington, Mass.) were used in the in vivo standard pharmacological test procedures. Human epidermoid carcinoma cells A-431 (American Type Culture Collection, Rockville, Md. #CRL-155) were grown in vitro as described above. A unit of 5xc3x97106 cells were injected subcutaneously into mice. When tumors attained a mass of between 100 and 150 mg, the mice were randomized into treatment groups (day zero). Mice were treated orally (PO) once a day on days 1 through 10 post staging with doses of either 40, 20, 10, 3 or 1 mg/kg/dose of the compound to be evaluated in 0.2% Klucel. Control animals received no drug. Tumor mass was determined every 7 days [(lengthxc3x97width2)/2] for 28 days post staging. Relative tumor growth (Mean tumor mass on day 7, 14, and 21 divided by the mean tumor mass on day zero) is determined for each treatment group. The % T/C (Tumor/Control) is determined by dividing the relative tumor growth of the treated group by the relative tumor growth of the placebo group and multiplying by 100. A compound is considered to be active if the % T/C is found to be=42%.
The ability of the compound of Example 9 to inhibit the growth of human epideimoid tumors (A431) in vivo is demonstrated below in TABLE 6.
As indicated by the results presented in TABLE 5, the compound of Example 9 is an effective inhibitor of tumor growth in vivo and is therfore useful for the treatment of cancer.
Based on the results obtained for representative compounds of this invention, the compounds of this invention are antineoplastic agents which are useful in treating, inhibiting the growth of, or eradicating neoplasms. In particular, the compounds of this invention are useful in treating, inhibiting the growth of, or eradicating neoplasms that express EGFR such as those of the breast, kidney, bladder, mouth, larynx, esophagus, stomach, colon, ovary, or lung. The compounds of this invention are also useful in treating, inhibiting the growth of, or eradicating neoplasms of the breast that express the receptor protein produced by the erbB2 (Her2) oncogene. Additionally, the compounds of this invention are useful in treating or inhibiting polycystic kidney disease and colonic polyps.
The compounds of this invention may formulated neat or may be combined with one or more pharmaceutically acceptable carriers for administration. For example, solvents, diluents and the like, and may be administered orally in such forms as tablets, capsules, dispersible powders, granules, or suspensions containing, for example, from about 0.05 to 5% of suspending agent, syrups containing, for example, from about 10 to 50% of sugar, and elixirs containing, for example, from about 20 to 50% ethanol, and the like, or parenterally in the form of sterile injectable solution or suspension containing from about 0.05 to 5% suspending agent in an isotonic medium. Such pharmaceutical preparations may contain, for example, from about 0.05 up to about 90% of the active ingredient in combination with the carrier, more usually between about 5% and 60% by weight.
The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration and the severity of the condition being treated. However, in general, satisfactory results are obtained when the compounds of the invention are administered at a daily dosage of from about 0.5 to about 1000 mg/kg of animal body weight, optionally given in divided doses two to four times a day, or in sustained release form. For most large mammals the total daily dosage is from about 1 to 1000 mg, preferably from about 2 to 500 mg. Dosage forms suitable for internal use comprise from about 0.5 to 1000 mg of the active compound in intimate admixture with a solid or liquid pharmaceutically acceptable callier. This dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The compounds of this invention may be administered orally as well as by intravenous, intramuscular, or subcutaneous routes. Solid catiers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, while liquid catTiers include sterile water, polyethylene glycols, non-ionic surfactants and edible oils such as corn, peanut and sesame oils, as are appropriate to the nature of the active ingredient and the particular form of administration desired. Adjuvants customarily employed in the preparation of pharmaceutical compositions may be advantageously included, such as flavoring agents, coloring agents, preserving agents, and antioxidants, for example, vitamin E, ascorbic acid, BHT and BHA.
The preferred pharmaceutical compositions from the standpoint of ease of preparation and administration are solid compositions, particularly tablets and hard-filled or liquid-filled capsules. Oral administration of the compounds is preferred.
In some cases it may be desirable to administer the compounds directly to the airways in the form of an aerosol.
The compounds of this invention may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparation contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
For the treatment of cancer, the compounds of this invention can be administered in combination with other antitumor substances or with radiation therapy. These other substances or radiation treatments can be given at the same or at different times as the compounds of this invention. These combined therapies may effect synergy and result in improved efficacy. For example, the compounds of this invention can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cisplatin or cyclophosamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, and antiestrogens such as tamoxifen.